Crystallized oxalate decarboxylase and methods of use

ABSTRACT

Oxalate decarboxylase crystals, including stabilized crystals, such as cross-linked crystals of oxalate decarboxylase, are disclosed. Methods to treat a disorder associated with elevated oxalate concentration using oxalate decarboxylase crystals are also disclosed. Additionally disclosed are methods of producing protein crystals.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. application Ser. No.60/834,933, filed on Aug. 2,2006 and U.S. application Ser. No.60/854,540, filed on Oct. 26, 2006. the contents of which are herebyincorporated by reference in their entireties.

BACKGROUND

Oxalic acid is a dicarboxylic acid of the formula HO₂C—CO₂H. Oxalic acidexists primarily as oxalate in biological organisms, which is the saltform of oxalic acid. Oxalate is found in foods, such as, e.g., spinach,rhubarb, strawberries, cranberries, nuts, cocoa, chocolate, peanutbutter, sorghum, and tea. Oxalate is also a metabolic end product inhumans and other mammals. It is excreted by the kidneys into the urine.When combined with calcium, oxalic acid produces an insoluble product,calcium oxalate, which is the most prevalent chemical compound found inkidney stones. Because mammals do not synthesize enzymes that degradeoxalate, oxalate levels in an individual are normally held in check byexcretion and low absorption of dietary oxalate. Elevated concentrationsof oxalate are associated with a variety of pathologies, such as primaryhyperoxaluria, enteric hyperoxaluria, and idiopathic hyperoxaluria.Leumann et al., Nephrol. Dial. Transplant. 14:2556-2558 (1999) andEarnest, Adv. Internal Medicine 24:407-427 (1979). Increased oxalate canbe caused by consuming too much oxalate from foods, by hyperabsorptionof oxalate from the intestinal tract, and by abnormalities of oxalateproduction. Hyperabsorption of oxalate in the colon and small intestinecan be associated with intestinal diseases, including hyperabsorptioncaused by diseases of bile acid and fat malabsorption; ileal resection;and, for example, by steatorrhea due to celiac disease, exocrinepancreatic insufficiency, intestinal disease, and liver disease.

Hyperoxaluria, or increased urinary oxalate excretion, is associatedwith a number of health problems related to the deposit of calciumoxalate in the kidney tissue (nephrocalcinosis) or urinary tract (e.g.,kidney stones, urolithiasis, and nephrolithiasis). Calcium oxalate mayalso be deposited in, e.g., the eyes, blood vessels, joints, bones,muscles, heart and other major organs, causing damage to the same. See.e.g., Leumann et al., J. Am. Soc. Nephrol. 12:1986 1993 (2001) andMonico et al., Kidney International 62:392 400 (2002). The effects ofincreased oxalate levels can appear in a variety of tissues. Forexample, deposits in small blood vessels cause painful skin ulcers thatdo not heal, deposits in bone marrow cause anemia, deposits in bonetissue cause fractures or affect growth in children, and calcium oxalatedeposits in the heart cause abnormalities of heart rhythm or poor heartfunction.

Existing methods to treat elevated oxalate levels are not alwayseffective and intensive dialysis and organ transplantation may berequired in many patients with primary hyperoxaluria. Existing therapiesfor various hyperoxalurias include high-dose pyridoxine, orthophosphate,magnesium, iron, aluminum, potassium citrate, cholestyramine, andglycosaminoglycan treatment, as well as regimes for adjusting diet andfluid intake, for dialysis, and for surgical intervention, such as renaland liver transplantation. These therapies (e.g., low-oxalate or low-fatdiet, pyridoxine, adequate calcium, and increased fluids), are onlypartially effective and they may have undesirable adverse side effects,such as the gastrointestinal effects of orthophosphate, magnesium, orcholestyramine supplementation and the risks of dialysis and surgery.Accordingly, methods that safely remove oxalate from the body areneeded. Moreover, methods that degrade oxalate to reduce oxalate levelsin a biological sample are advantageous over a therapy, for example,that solely blocks absorption or increased clearance of oxalate.

SUMMARY

The invention relates to crystals of oxalate decarboxylase (“OXDC”) andcross-linked forms thereof (“CLEC”) and their uses to treatoxalate-associated disorders, e.g., hyperoxaluria. In one embodiment,crystalline oxalate decarboxylase can be administered to a mammal, e.g.,orally or directly to the stomach, to reduce oxalate levels and/or toreduce damage caused by calcium oxalate deposits in the mammal.Additionally disclosed are methods of producing protein crystals fromcell extracts. Compositions, e.g., pharmaceutical compositions,including the crystals of oxalate decarboxylase (“OXDC”) andcross-linked forms thereof (“CLEC”) are also disclosed.

In one aspect, the invention provides cross-linked oxalate decarboxylasecrystals. The cross-linking agent can be multifunctional, and in certainembodiments, the agent is a bifunctional agent, such as glutaraldehyde.In certain embodiments, the oxalate decarboxylase crystals arecross-linked with glataraldahyde at a concentration that does notsubstantially alter enzyme activity, e.g., at a concentration of atleast about 0.02% (w/v), in embodiments, the level of cross-linking ofthe oxalate decarboxylase crystal is equivalent to that produced bytreatment with 0.02% (w/v) glutaraldehyde. The level of cross-linkingcan be determined by methods known in the art or disclosed herein, e.g.,determining the level of protein leaching, e.g., as disclosed inExamples 10-11.

The invention farther provides oxalate decarboxylate crystals, e.g.,oxalate decarboxylate crystals that have a higher activity, e.g., atleast about 100%, 200%, 300%, 400% or 500%, compared to the solubleoxalate decarboxylate.

The invention further provides a stabilized, e.g., cross-linked, oxalatedecarboxylase crystal, wherein said stabilized crystal retains anactivity and/or stability, in acidic conditions at least 2-, 3-foldhigher than the activity and/or stability retained by a soluble oxalatedecarboxylase in similar acidic conditions (e.g., an acidic pH of about2 to 3). In embodiments, the stabilized oxalate decarboxylase crystal isat least 200%, 300%, 400% more active and/or stable than a solubleoxalate decarboxylase in acidic conditions.

The invention farther provides a stabilized, e.g., cross-linked, oxalatedecarboxylase crystal, wherein said stabilized crystal retains anactivity and/or stability, in the presence of a protease, at least 2-,3-fold higher than the activity and/or stability retained by a solubleoxalate decarboxylase in similar conditions. In embodiments, thestabilized oxalate decarboxylase crystal is at least 200%, 300%, 400%more active and/or stable than a soluble oxalate decarboxylase in thepresence of a protease. The protease can be chosen from one or more of,pepsin, chymotrypsin or pancreatin. In embodiments, the activity of thestabilized or soluble oxalate decarboxylase is measured after exposingthe stabilized crystal or soluble oxalate decarboxylase to acidicconditions and/or a protease for a predetermined length of time, e.g.,at least one, two, three, four or five hours as described in theExamples herein).

In a related aspect, the invention features a cross-linked, oxalatedecarboxylase crystal which is substantially active and stable invariable pH conditions (e.g., about pH 2.5 or 3 to 7.5 or 8.5), and/orin the presence of a protease, e.g., a protease can be chosen from oneor more of, e.g., pepsin, chymotrypsin or pancreatin. In embodiments,the cross-linked crystal retains an activity at least 2-, 3-fold higherthan the activity retained by a soluble oxalate decarboxylase in acidicconditions (e.g., an acidic pH of about 2 to 3) and in the presence of aprotease, as described herein. In other embodiments, the stabilizedoxalate decarboxylase crystal is at least 200%, 300%, 400% more stablethan a soluble oxalate decarboxylase in acidic conditions (e.g., anacidic pH of about 2 to 3) and in the presence of a protease, asdescribed herein.

Compositions, e.g., pharmaceutical compositions, that include thecrystals and/or the cross-linked oxalate decarboxylase crystals asdescribed herein are also within the scope of the invention.

In some embodiments, the crystals include oxalate decarboxylase having asequence identical or substantially identical to an oxalatedecarboxylase sequence found in a natural source, such as a plant,bacterium and fungus, in particular from Bacillus subtilis, Collybiavelulipes or Flammulina velutipes, Aspergillus niger, Pseudomonas sp.Synechocystis sp. Streptococcus mutans, Trametes hirsute, Sclerotiniasclerotiorum, T. versicolor, Postia placenta, Myrothecium verrucaria,Agaricus bisporus, Methylobactetium extorquens, Pseudomonas oxalaticus,Rafstonia eutropha, Cupriavidus oxalaticus, Wautersia sp.,Oxalicibactenum flavum, Ammoniiphilus oxalaticus, Vibrio oxalaticus, A.oxalativorans, Varlovorax paradoxus, Xanthobacter autotrophicus,Aspergillus sp., Penicillium sp., and Mucor species. In otherembodiments, the oxalate decarboxylase is recombinantly produced.

In one aspect, the invention provides a method of reducing oxalateconcentration in a subject by administering a composition, e.g., apharmaceutical composition, that includes oxalate decarboxylasecrystals, e.g., cross-linked oxalate decarboxylase crystals, asdisclosed herein. In one embodiment, the oxalate decarboxylase crystalsare stabilized by a cross-linking agent, such as glutaraldchyde.Administration of the composition can cause a reduction of oxalateconcentration by at least 10%. at least 20%, at least 30%, or at least40% or more. In some embodiments, the composition is administered orallyor via an extracorporeal device. In one embodiment, the extracorporealdevice is a catheter, e.g., a catheter coated with oxalate decarboxylasecrystals. In other embodiments, the composition is administered as asuspension, dry powder, capsule, or tablet. In one embodiment, themethod of reducing oxalate concentration in a mammal includes a step ofassaying the oxalate concentration in a biological sample of the mammal,such as a urine, blood, plasma, or serum sample.

In another aspect, the invention provides a method of treating,prevolting, and/or slowing the progression of a disorder associated withelevated oxalate concentrations in a mammal by administering oxalatedecarboxylase crystals and/or stabilized, e.g., cross-linked, oxalatedecarboxylase crystals to the mammal. In one embodiment, the disorderassociated with elevated oxalate concentration is a kidney disorder,joint disorder, eye disorder, liver disorder, gastrointestinal disorder,or pancreatic disorder, in certain embodiments, the disorder is primaryhyperoxaluria, enteric hyperoxaluria, idiopathic hyperoxaluria, ethyleneglycol poisoning, cystic fibrosis, inflammatory bowel disease,urolithiasis, nephrolithiasis, chronic kidney disease, hemodialysis, andgastrointestinal bypass.

In another aspect, the invention provides a composition, e.g., apharmaceutical composition, that includes oxalate decarboxylasecrystals, e.g., cross-linked oxalate decarboxylase crystals (e.g., thecrystals and/or cross-linked crystals, as disclosed herein).

In yet another aspect, the invention provides a method of treating amammal by administering an effective amount of a pharmaceuticalcomposition that includes oxalate decarboxylase crystals, e.g.,cross-linked oxalate decarboxylase crystals (e.g., the crystals and/orcross-linked crystals, as disclosed herein).

In another aspect, the invention provides methods of producing proteincrystals, e.g., enzyme crystals (e.g., oxalate decarboxylase crystals),that include: providing a preparation of cell extract orpellets/precipitate/solution containing the protein, and crystallizingthe protein from the preparations. In embodiments, the methods includeone or more of: culturing a prokaryotic host cell culture expressing theprotein; obtaining, a preparation of pellets or extracts containingdesired. protein; solubilizing the preparation of pellets; allowing,protein crystals to form, and/or further stabilizing the crystals bycross-linking. Typically, the protein is expressed recombinantly. Inembodiments, the pellet preparation includes inclusion bodies.

In embodiments, the solubilization step includes adding to thepreparation of pellets one or more of: a solution comprising a milddenaturant concentration (e.g., urea or guanidine hydrochloride at aconcentration, e.g., about 1 M to about 3M); a solution comprising ahigh salt concentration, e.g., a salt chosen from one or more of sodiumchloride, potassium chloride, calcium chloride, or other salts at aconcentration of, e.g., about 0.3 to about 0.8 M; a solution comprisinga mild denaturant concentration under basic conditions, e.g., at a pH ofabout 9 to about 12; or a solution comprising a high denaturantconcentration, e.g., about 4 M to about 8 M urea or guanidinehydrochloride.

In embodiments, the purifying step includes removing debris from thepellets, e.g., by separating, e.g., by one or more spinning orcentrifugation steps, the solubilized preparation of pellets and/orcollecting the supernatant. The purifying step may, optionally, furtherinclude passing the solubilized preparation of pellets through ionexchange chromatography, and/or filtering the solubilized preparation.

In embodiments, the crystallizing step includes concentrating thepurified protein, thereby forming crystallized protein. In embodiments,the crystallized protein is obtained from the supernatant collectedafter the separation step, e.g., after one or more spinning orcentrifugation steps. The crystallization step can additionally includecontacting the crystallized protein with a cross-linking agent, e.g.,across-linking agent disclosed herein (e.g., glutaraldehyde). Theconcentration of crosslinking agent used can be in the range of 0.01% to20% w/v; typically, 0.02% to 10% w/v; more typically, 0.02%, 0.5% or 1%w/v.

In embodiments, the yield of the protein in the pellet preparation is atleast about 50%, 60%, 70%, 80% of the specific protein found in the cellpreparation from which the pellet preparation is obtained. In otherembodiments, the yield of the solubilized protein is at least about 90%,95% or higher of that found in the pellet preparation. In yet otherembodiments, the yield of the crystallized protein is at least about50%, 60%, 70%, 80% of that found in the pellet preparation.

The invention further provides protein crystals, e.g., enzyme crystals(e.g., oxalate decarboxylase crystals) produced by the methods disclosedherein.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below.

DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing the pH activity profile of soluble oxalatedecarboxylase (“Soluble”), oxalate decarboxylase crystals (“Crystal”),and cross-linked oxalate decarboxylase crystals (“CLEC”).

FIG. 2 is a bar graph depicting urinary oxalate levels in Sprague Dawleyrats after administration of OXDC-CLEC. Each bar represents a mean ±SE(standard error). The asterisks indicate a significant differencebetween the control group and three treatment groups at p<0.05 at thetime point, using a two tailed Student t-test. FIG. 3 is a bar graphdepicting the effect of OXDC-CLEC on reduction of urinary oxalate levelsin ethylene glycol-challenged AGT1 knock-out (KO) mice. Each barrepresents the mean SE. Asterisks indicate a significant differencebetween the control group and three treatment groups at p<0.05 at thetime point, calculated using a two tailed Student's t-test.

FIG. 4 is a bar graph depicting the effect of OXDC-CLEC on creatinineclearance in ethylene glycol-challenged AGT1 KO mice. Each barrepresents the mean ±SE. Asterisks indicate a significant differencebetween the control group and 80 mg treatment group at p<0.05 by a twotailed Student's t-test.

FIGS. 5A-5C are images showing the prevention of calcium oxalatedeposits on kidney parenchyma following treatment with OXDC-CLEC. FIG.5A is a section of kidney parenchyma from EG challenged mice treatedwith 80 mg OXDC-CLEC. FIGS. 5B and 5C arc section of kidney parenchymafrom the control group. The section if FIG. 5B demonstrates moderatenephrocalcinosis and the section in FIG. 5C demonstrates severenephrocalcinosis. The dark patches are calcium oxalate deposits (exampleindicated by a white arrow), and the light patches are areas withinterstitial fibrosis (examples indicated by gray arrows).

FIG. 6 is a Kaplan-Meier survival plot comparing the survival times ofEG-challenged mice treated with three different doses of OXDC-CLEC or avehicle control.

FIG. 7 is a graph depicting the stability of soluble oxalatedecarboxylase (“Sol”), oxalate decarboxylase crystals (“XTAL”), andcross-linked oxalate decarboxylase crystals (“CLEC”) at low pH at thetime intervals indicated.

FIG. 8 is a graph depicting the stability of soluble oxalatedecarboxylase (“Sol”), oxalate decarboxylase crystals (“XTAL”), andcross-linked oxalate decarboxylase crystals (“CLEC”) in the presence ofpepsin at pH 3.0 at the time intervals indicated.

FIG. 9 is a graph depicting the stability of soluble oxalatedecarboxylase (“Sol”), oxalate decarboxylase crystals (“XTAL”) andcross-linked oxalate decarboxylase crystals (“CLEC”) in the presence ofchymotrypsin at pH 7.5 at the time intervals indicated.

FIG. 10 is a graph depicting the stability of soluble oxalatedecarboxylase (“Sol”), oxalate decarboxylase crystals (“XTAL”), andcross-linked oxalate decarboxylase crystals (“CLEC”) in the presence ofsimulated intestinal juice with pancreatin at pH 6.8 at the timeintervals indicated.

DETAILED DESCRIPTION

The present invention is based, in part, on the discovery thatadministering crystals of oxalate decarboxylase (OXDC) can reduce thesymptoms of hyperoxaluria in a mammal. Methods of administering OXDCcrystals to treat various oxalate-related disorders are describedherein. Additionally, OXDC crystals and cross-linked crystals (CLECs)are provided, as are compositions comprising and using the same.Additionally disclosed are methods of producing large quantities ofprotein crystals from cell extracts of a prokaryotic host cell.

Definitions. In order that the present invention may be more readilyunderstood, certain terms are first defined. Additional definitions areset forth throughout the detailed description.

As used herein, a “biological sample” is biological material collectedfrom cells, tissues, organs, or organisms, for example, to detect ananalyte. Exemplary biological samples include a fluid, cell, or tissuesample. Biological fluids include, for example, serum, blood, plasma,saliva, urine, or sweat. Cell or tissue samples include biopsy, tissue,cell suspension, or other specimens and samples, such as clinicalsamples.

A “crystal” is one form of the solid state of matter, comprising atomsarranged in a pattern that repeats periodically in three dimensions(see, e.g., Barret, Structure of Metals, 2^(nd) ed., McGraw-Hill, NewYork (1952)). A crystal form of a polypeptide, for example, is distinctfrom a second form the amorphous solid state. Crystals displaycharacteristic features including shape, lattice structure, percentsolvent, and optical properties, such as, e.g., refractive index.

An “extracorporeal device” is a structure that is not within the bodyfor bringing a body fluid in contact with OXDC crystals in the treatmentof an individual. Preferably, an extracorporeal device is a device usedfor dialysis, including kidney dialysis, a device for continuousarteriovenous hemofiltration, an extracorporeal membrane oxygenator, orother device used to filter waste products from the bloodstream.Similarly, components of devices to filter waste products areencompassed by the term, including a tube, a porous material, or amembrane, for example. In particular, an extracorporeal device may be adialysis device. It may also be a membrane of a dialysis device.

A “functional fragment” of OXDC is a portion of an OXDC polypeptide thatretains one or more biological activities of OXDC, such as the abilityto catalyze the decarboxylation of oxalate. As used herein, a functionalfragment may comprise terminal truncations from one or both termini,unless otherwise specified. For example, a functional fragment may have1, 2, 4, 5, 6, 8, 10, 12, 15, or 20 or more residues omitted from theamino and/or carboxyl terminus of an OXDC polypeptide. Preferably, thetruncations are not more than 20 amino acids from one or both termini. Afunctional fragment may optionally be linked to one or more heterologoussequences.

The term “individual” or “subject” refers to any mammal, including butnot limited to, any animal classified as such, including humans, nonhuman primates, primates, baboons, chimpanzees, monkeys, rodents (e.g.,mice, rats), rabbits, cats, dogs, horses, cows, sheep, goats, pigs, etc.

The term “isolated” refers to a molecule that is substantially free ofits natural environment. For instance, an isolated protein issubstantially free of cellular material or other proteins from the cellor tissue source from which it is derived. The term refers topreparations where the isolated protein is sufficiently pure to beadministered as a therapeutic composition, or at least 70% to 80% (w/w)pure, more preferably, at least 80% 90% (w/w) pure, even morepreferably, 90 to 95% pure; and, most preferably, at least 95%, 96%,97%, 98%, 99%, 99.5%, 99.8% or 100% (w/w) pure.

As used herein, the term “about” refers to up to 10% of the valuequalified by this term. For example, about 50 mM refers to 50 mM±mM;about 4% refers to 4±0.4%.

As used herein, “oxalate-associated disorder” refers to a disease ordisorder associated with pathologic levels of oxalic acid or oxalate,including, but not limited to hyperoxaluria, primarily hyperoxaluria,enteric hyperoxaluria, idiopathic hyperoxaluria, ethylene glycol(oxalate) poisoning, idiopathic urinary stone disease, renal failure(including progressive, chronic, or end-stage renal failure),steatorrhoea, malabsorption, ileal disease, vulvodynia, cardiacconductance disorders, inflammatory bowel disease, cystic fibrosis,exocrine pancreatic insufficiency, Crohn's disease, ulcerative colitis,nephrocalcinosis, urolithiasis, and nephrolithiasis. Such conditions anddisorders may optionally be acute or chronic. Oxalate-associateddisorders associated with kidneys, bone, liver, gastrointestinal tract,and pancreas are known in the art. Further, it is well known thatcalcium oxalate can deposit in a wide variety of tissues including, butnot limited to, the eyes, blood vessels, joints, bones, muscles, heart,and other major organs leading to a number of oxalate-associateddisorders.

“Oxalic acid” exists predominantly in its salt form, oxalate (as saltsof the corresponding conjugate base), at the pH of urine and intestinalfluid (pK_(a1)1.23, pK_(a2)=4.19). Earnest, Adv Internal Medicine 24:407427 (1979), The terms “oxalic acid” and “oxalate” are usedinterchangeably throughout this disclosure. Oxalate salts comprisinglithium, sodium, potassium, and iron (II) are soluble, but calciumoxalate is typically very poorly soluble in water (for example,dissolving only to about 0.58 mg/100 ml at 18° C. Earnest, Adv. InternalMedicine 24:407 427 (1979)). Oxalic acid from food is also referred toas dietary oxalate. Oxalate that is produced by metabolic processes isreferred to as endogenous oxalate. Circulating oxalate is the oxalatepresent in a circulating body fluid, such as blood.

The terms “therapeutically effective dose,” or “therapeuticallyeffective amount,” refer to that amount of a compound that results inprevention, delay of onset of symptoms, or amelioration of symptoms ofan oxalate-related condition, including hyperoxaluria, such as primaryhyperoxaluria or enteric hyperoxaluria. A therapeutically effectiveamount will, for example, be sufficient to treat, prevent, reduce theseverity, delay the onset, and/or reduce the risk of occurrence of oneor more symptoms of a disorder associated with elevated oxalateconcentrations. The effective amount can be determined by methods wellknown in the art and as described in subsequent sections of thisdescription.

The terms “treatment,” “therapeutic method,” and their cognates refer totreatment of an existing disorder and/or prophylactic/preventativemeasures. Those in need of treatment may include individuals alreadyhaving a particular medical disorder, as well as those at risk orhaving, or who may ultimately acquire the disorder. The need fortreatment is assessed, for example, by the presence of one or more riskfactors associated with the development of a disorder, the presence orprogression of a disorder, or likely receptiveness to treatment of asubject having the disorder. Treatment may include slowing or reversingthe progression of a disorder.

Oxalate decarboxylase. As used herein, oxalate decarboxylase (OXDC) (EC4.1.1.2) refers to an oxalate carboxy-lyase enzyme. Oxalatedecarboxylases are a group of enzymes known in the art capable ofcatalyzing the molecular oxygen (O₂) independent oxidation of oxalate tocarbon dioxide and formate according to the following reaction:

HO₂C—CO₂H→1 CO₂+HCOOH

Isoforms of oxalate decarboxylase, and glycoforms of those isoforms, areincluded within this definition. OXDC from plants, bacteria and fungiare encompassed by the term, including the true oxalate decarboxylasesfrom bacteria and fungi, such as Bacillus subtilis, Collybia velutipesor Flammulina velutipes, Aspergillus niger, Pseudomonas sp.,Synechocystis sp., Streptococcus mutans, Trametes hirsute, Selerotiniasclerotiorum, T. versicolor, Postia placenta, Mywrothecium verrucaria,Agaricus bisporus, Methylobacterium extorquens, Pseudomonas oxalaficus,Kalstonia eutropha, Cupriavidus oxalaticus, Wautersia sp.,Oxalicibacterium flavum, Ammoniiphilus oxalaticus, Vibrio oxalaticus, A.oxalativorans. Variovorax paradoxus, Xanthobacter autotrophicus,Aspergillus sp., Penicillium sp., and Mucor species. Optionally, theOXDC will be additionally dependent on coenzyme A, such as OXDC fromorganisms in the intestinal tract. In certain circumstances, OXDC is asoluble hexameric protein. Oxalate decarboxylases are produced by higherplants, bacteria, and fungi and have oxalate carboxy-lyase enzymaticactivity. Oxalate decarboxylases include those produced by Bacillussubtilis, Collybia velutipes or Flammulina velutipes, Aspergillus niger,Pseudomonas sp., Synechocystis sp., Streptococcus mutans, Trameteshirsute, Sclerotinia sclerotiorum, T. versicolor, Postia placenta,Myrothecium verrucaria, Agaricus bisporus, Methylobacterium extorquens,Pseudomonas oxalaticus, Rahtonia eutropha, Cupriavidus oxalaticus,Wautersia sp., Oxalicibacterium flavum, Ammoniiphilus oxalaticus, Vibriooxalaticus, A. oxalativorans, Variovorax paradoxus, Xanthobacterautotrophicus, Aspergillus sp., Penicillium sp., and Mucor species aregenerally identified as cupin type OXDCs. The cupin-like proteins are alarge class of proteins sharing certain structural features. OXDCs, suchas G-OXDCs from Collybia sp., are active as, for example, hexamericglycoproteins.

Oxalate decarboxylases used to prepare the crystals, and which are usedin methods described herein, may be isolated, for example, from anatural source, or may be derived from a natural source. As used herein,the term “derived from” means having an amino acid or nucleic acidsequence that naturally occurs in the source. For example, oxalatedecarboxylase derived from Bacillus subtilis will comprise a primarysequence of a Bacillus subtilis oxalate decarboxylase protein, or willbe encoded by a nucleic acid comprising a sequence found in Bacillussubtilis that encodes an oxalate decarboxylase or a degenerate thereof.A protein or nucleic acid derived from a source encompasses moleculesthat are isolated from the source, recombinantly produced, and/orchemically synthesized or modified. The crystals provided herein may beformed from polypeptides comprising amino acid sequences of OXDC, or afunctional fragment of OXDC that retains oxalate degrading activity.Preferably, the OXDC retains at least one functional characteristic of anaturally occurring OXDC, e.g., retains one or more of the ability tocatalyze degradation of oxalate, ability to multimerize, and/ormanganese requirement.

Isolated Oxalate Decarboxylase. Oxalate decarboxylases have beenpreviously isolated and are thus available from many sources, includingBacillus subtilis, Collybia velutipes or Flammulina velutipes,Aspergillus niger, Pseudomonas sp., Synechocystis sp., Streptococcusmutans, Trametes hirsute, Sclerotinia sclerotiorum, T. versicolor,Postia placenta, Myrothecium verrucaria, Agaricus bisporus,Methylobacterium extorquens, Pseudomonas oxalaticus, Ralstonia eutropha,Cupriavidus oxalaticus, Wautersia sp., Oxalicibacterium flavum,Ammoniiphilus oxalaticus, Vibrio oxalaticus, A. oxalativorans,Variovorax paradoxus, Xanthobacter autotrophicus, Aspergillus sp.,Penicillium sp., and Mucor species. OXDC may also be purchased fromcommercial purveyors, such as, e.g.. Sigma. Methods to isolate OXDC froma natural source are previously described, for example, in the followingreferences: Tanner et al., The Journal of Biological Chemistry.47:43627-43634. (2001); Dashek, W. V. and Micales. J. A., Methods inplant biochemistry and molecular biology. Boca Raton, Fla.: CRC Press.5:49-71. (1907); Magro et al., FEMS Microbiology Letters. 49: 49-52.(1988); Anand et al., Biochemistry. 41: 7659-7669. (2002); and Tannerand Bornemann, S. Journal of Bacteriology. 182; 5271-5273 (2000). Theseisolated oxalate decarboxylases may be used to form the crystals andmethods described herein.

Recombinant Oxalate Decarboxylase. Alternatively, recombinant OXDCs maybe used to form the crystals and methods provided herein. In someinstances, recombinant OXDCs encompass or arc encoded by sequences froma naturally occurring OXDC sequence. Further, OXDCs comprising an aminoacid sequence that is homologous or substantially identical to anaturally occurring sequence are herein described. Also, OXDCs encodedby a nucleic acid that is homologous or substantially identical to anaturally occurring OXDC-encoding nucleic acid are provided and may becrystallized and/or administered as described herein.

Polypeptides referred to herein as “recombinant” are polypeptides whichhave been produced by recombinant DNA methodology, including those thatare generated by procedures which rely upon a method of artificialrecombination, such as the polymerase chain reaction (PCR) and/orcloning into a vector using restriction enzymes. “Recombinant”polypeptides arc also polypeptides having altered expression, such as anaturally occurring polypeptide with recombinantly modified expressionin a cell, such as a host cell.

In one embodiment, OXDC is recombinantly produced from a nucleic acidthat is homologous to a Bacillus subtilis or Coltybia velutipes OXDCnucleic acid sequence, and sometimes it is modified, e.g., to increaseor optimize recombinant production in a heterologous host. An example ofsuch a modified sequence is provided in SEQ ID NO: 1 (nucleic acid),which includes the nucleic acid sequence of the open reading frame ofCollybia velutipes OXDC, for expression in Candida boidinii. The OXDCsequence has been modified to reduce its GC. content, is linked to an αMating Factor secretion signal sequence, and is flanked by engineeredrestriction endonuclease cleavage sites. In another embodiment, OXDC isrecombinantly produced from SEQ ID NO:2, or the unmodified Bacillussubtilis OXDC nucleic acid sequence that is available at GenBankAccession No: Z99120. The amino acid sequence encoded by SEQ ID NO:2 isprovided as SEQ ID NO:3.

OXDC polypeptides useful for forming OXDC crystals may be expressed in ahost cell, such as a host cell comprising a nucleic acid construct thatincludes a coding sequence for an OXDC polypeptide or a functionalfragment thereof. A suitable host cell for expression of OXDC may beyeast, bacteria, fungus, insect, plant, or mammalian cell, for example,or transgenic plants, transgenic animals or a cell-free system.Preferably, a host cell is capable of glycosylating the OXDC polypeptideif necessary, capable of disulfide linkages, capable of secreting theOXDC and/or capable of supporting multimerization of OXDC polypeptides.Preferred host cells include, but are not limited to E. coli (includingE. coli Origami B and E. coli BL21), Pichia pastoris, Saccharomycescerevisiae, Schizosaccharomyces pombe, Bacillus subtilis, Aspergillus,Sf9 cells. Chinese hamster ovary (CHO), 293 cells (human embryonickidney), and other human cells. Also transgenic plants, transgenicanimals including pig, cow, goat, horse, chicken, and rabbit aresuitable hosts for production of OXDC.

For recombinant production of OXDC, a host or host cell should comprisea construct in the form of a plasmid, vector, phagemid, or transcriptionor expression cassette that comprises at least one nucleic acid encodingan OXDC or a functional fragment thereof. A variety of constructs areavailable, including constructs which are maintained in single copy ormultiple copy, or which become integrated into the host cell chromosome.Many recombinant expression systems, components, and reagents forrecombinant expression are commercially available, for example fromInvitrogen Corporation (Carlsbad, Calif.); U.S. Biological (Swampscott,Mass.); BD Biosciences Pharmingen (San Diego, Calif.); Novagen (Madison,Wis.); Stratagene (La Jolla, Calif.); and Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH (DSMZ). (Braunschweig, Germany).

Recombinant expression of OXDC is optionally controlled by aheterologous promoter, including a constitutive and/or induciblepromoter. Promoters such as, e.g., T7, the alcohol oxidase (AOX)promoter, the dihydroxy-acetone synthase (DAS) promoters, the GaI 1,10promoter, the phosphoglycerate kinase promoter, theglyceraldehyde-3-phosphate dehydrogenase promoter, alcohol dehydrogenasepromoter, copper metallothioncin (CUP1) promoter, acid phosphatasepromoter, CMV and promoters polyhedrin are also appropriate. Theparticular promoter is selected based on the host or host cell. Inaddition, promoters that are inducible by methanol, copper sulfate,galactose, by low phosphate, by alcohol, e.g., ethanol, for example, mayalso be used und are well known in the art.

A nucleic acid that encodes OXDC may optionally comprise heterologoussequences. For example, a secretion sequence is included at theN-terminus of an OXDC polypeptide in some embodiments. Signal sequencessuch as those from α Mating Factor, BGL2, yeast acid phosphatase (PHO),xylanase. alpha amylase, from other yeast secreted proteins, andsecretion signal peptides derived from other species that are capable ofdirecting secretion from the host cell may be useful. Similarly otherheterologous sequences such as linkers (e.g., comprising a cleavage orrestriction endonuclease site) and one or more expression controlelements, an enhancer, a terminator, a leader sequence, and one or moretranslation signals are within the scope of this description. Thesesequences may optionally be included in a construct and/or linked to thenucleic acid that encodes OXDC. Unless otherwise specified, “linked”sequences can be directly or indirectly associated with one another.

Similarly, an epitope or affinity tag such as Histidine, HA(hemagglutinin peptide), maltose binding protein, AviTag®, FLAG, orglutatthione-S-transferase may be optionally linked to the OXDCpolypeptide. A tag may be optionally cleavable from the OXDC after it isproduced or purified. A skilled artisan can readily select appropriateheterologous sequences, for example, match host cell, construct,promoter, and/or secretion signal sequence.

OXDC homologs or variants differ from an OXDC reference Sequence by oneor more residues. Structurally similar amino acids can be substitutedfor some of the specified amino acids, for example. Structurally similaramino acids include: (I, L and V); (F and Y); (K and R); (Q and N), (Dand E); and (G and A). Deletion, addition, or substitution of aminoacids is also encompassed by the OXDC homologs described herein. Suchhomologs and variants include (i) polymorphic variants and natural orartificial mutants, (ii) Modified polypeptides in which one or moreresidues is modified, and (iii) mutants comprising one or more modifiedresidues.

OXDC polypeptide or nucleic acid is “homologous” (or is a “homolog”) ifit is at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%,99%, or 100% identical to a reference sequence. If the homolog is notidentical to the reference sequence, it is a “variant.” A homolog is“substantially identical” to a reference OXDC sequence if the nucleotideor amino acid sequence of the homolog differs from the referencesequence (e.g. , truncation, deletion, substitution, or addition) by nomore than 1, 2, 3, 4, 5, 8, 10, 20, or 50 residues, and retains (orencodes a polypeptide that retains) the ability to catalyze thedegradation of oxalate. Fragments of all oxalate decarboxylase may behomologs, including variants and/or substantially identical sequences.By way of example, homologs may be derived from various sources of OXDC,or they may be derived from or related to a reference sequence bytruncation, deletion, substitution, or addition mutation. Percentidentity between two nucleotide or amino acid sequences may bedetermined by standard alignment algorithms such as, for example, BasicLocal Alignment Tool (BLAST) described in Altschul et al.,J. Mol. Biol.,215:403 410 (1990), the algorithm of Needleman et al., J. Mol. Biol.,48: 444 453 (1970), or the algorithm of Meyers et al., Compute. Appl.Biosci. 4: 11 17 (1988). Such algorithms are incorporated into theBLASTN, BLASTP and “BLAST 2 Sequences” programs (reviewed in McGinnisand Madden, Nucleic. Acids Res. 32:W20-W25, 2004). When utilizing suchprograms, the default parameters can be used. For example, fornucleotide sequences the following settings can be used for “BLAST 2Sequences”: program BLASTN, reward for match 2, penalty for mismatch 2,open gap and extension gap penalties 5 and 2 respectively, gap x_dropoff50, expect 10, word size 11, filter ON. For amino acid sequences thefollowing settings can be used tor “BLAST 2 Sequences”: program BLASTP,matrix BLOSUM62, open gap and extension gap penalties 11 and 1respectively, gap x_dropoff 50, expect 10, word size 3, filter ON. Theamino acid and nucleic acid sequences for OXDCs that are appropriate toform the crystals described herein, may include homologous, variant, orsubstantially identical sequences. Purification of OxalateDecarboxylase. Oxalate decarboxylase proteins or polypeptides may bepurified from the source, such as a natural or recombinant source, priorto crystallization. A polypeptide that is referred to herein as“isolated” is a polypeptide that is substantially free of its naturalenvironment, such as proteins, lipids, and/or nucleic acids of theirsource of origin (e.g., cells, tissue (i.e., plant tissue), or fluid ormedium (in the case of a secreted polypeptide)). Isolated polypeptidesinclude those obtained by methods described herein or other suitablemethods, and include polypeptides that are substantially pure oressentially pure, and polypeptides produced by chemical synthesis, byrecombinant production, or by combinations of biological and chemicalmethods. Optionally, an isolated protein has undergone furtherprocessing after its production, such as by purification steps.

Purification may comprise buffer exchange and chromatographic steps.Optionally, a concentration step may be used, e.g., by dialysis,chromatofocusing chromatography, and/or associated with buffer exchange.In certain instances, cation or anion exchange chromatography is usedfor purification, including Q-sepharose, DEAE sepharose, DE52,sulfopropyl Sepharose chromatography or a CM52 or similar cationexchange column. Buffer exchange optionally precedes chromatographicseparation, and may be performed by tangential flow filtration such asdiafiltration. In certain preparations, OXDC is at least 70%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% pure.

Purification in gram-scale runs is appropriate to prepare OXDC, andprocedures are optimized for efficient, inexpensive, manufacturing-scaleOXDC purification. For example, purification of at least 0, 5, 1, 2, 5,10, 20, 50, 100, 500, or 1000 grams or more of OXDC in a purificationprocedure is provided. In one exemplary procedure, tangential flowfiltration of starting samples of at least 10L, 50L, 100L, 500L, 1000Lor more is provided, allowing buffer exchange and precipitation ofcontaminant proteins. A single Q-sepharose column is optionally used forpurification of OXDC.

Crystallization of purified OXDC may also remove contaminants, forexample to further purify OXDC preparations. For example, OXDCcystallized as described in Examples 2-6, has reduced levels of lowmolecular weight contaminants, as compared to soluble purified OXDC. Insome aspects, contaminants having a measured mass (by matrix assistedlaser desorption ionization mass spectroscopy (MALDI-MS)) of 0-10 KDa,1-10 KDa, 0.5-5 KDa, or 2-5 KDa are selectively excluded from thecrystal form. For example, contaminants having measured masses ofapproximately 2.5, 3.0, 3.7, 3.8, 4.0. 4.2, or 5.0 KDa can besubstantially removed by crystallization. Purification bycrystallization may also be done using, e.g. crude oxalate decarboxylasecontaining fermentation media.

Crystallization of Oxalate Decarboxylase. Oxalate decarboxylase crystalscan be prepared using an (OXDC polypeptide, such as a hexamer, asdescribed above (see Anand et al., Biochemistry 41:7659-7669 (2002)).Vapor diffusion (such as, e.g., hanging drop and sitting drop methods),and batch methods of crystallization, for example, can be used. Oxalatedecarboxylase crystals may be grown by controlled crystallization of theprotein out of an aqueous solution or an aqueous solution that includesorganic solvents. Conditions to be controlled include the rate ofevaporation of solvent, the presence of appropriate co-solutes andbuffers, pH, and temperature, for example.

For therapeutic administration, such as to treat a condition or disorderrelated to oxalate levels, a variety of OXDC crystal sizes areappropriate. In certain embodiments, crystals of less than about 500 μmaverage dimension are administered. Oxalate decarboxylase crystals withan average, maximal, or minimal dimension (for example) that is about0.01, 0.1, 1, 5, 10, 25, 50, 100, 200, 300, 400, 500, or 1000 μm inlength are also provided. Microcrystalline showers are also suitable.

Ranges are appropriate and would be apparent to the skilled artisan. Forexample, the protein crystals may have a longest dimension between about0.01 μm and about 500 μm, alternatively, between 0.1 μm and about 50 μm,in a particular embodiment, the longest dimension ranges from about 0.1μm to about 10 μm. Crystals may also have a shape chosen from spheres,needles, rods, plates, such as hexagons and squares, rhomboids, cubes,bipyramids and prisms. In illustrative embodiments, the crystals arecubes having a longest dimension of less than 5 μm.

In general, crystals are produced by combining the protein to becrystallized with an appropriate aqueous solvent or aqueous solventcontaining appropriate crystallization agents, such as salts or organicsolvents. The solvent is combined with the protein and optionallysubjected to agitation at a temperature determined experimentally to beappropriate for the induction of crystallization and acceptable for themaintenance of protein activity and stability. The solvent canoptionally include co-solutes, such as monovalent or divalent cations,co-factors or chaotropes, as well as buffer species to control pH. Theneed for co-solutes and their concentrations are determinedexperimentally to facilitate crystallization. In an industrial scaleprocess, the controlled precipitation leading to crystallization can becarried out by the combination of protein, precipitant, co-solutes and,optionally, buffers in a batch process, for example. Alternativelaboratory crystallization methods and conditions, such as dialysis orvapor diffusion, can be adopted (McPherson, et al., Methods Enzymol.114:112-20 (1985) and Gilliland, Crystal Growth 90:51-59 (1998)).Occasionally, incompatibility between the cross-linking agent and thecrystallization medium might require changing the buffers (solvent)prior to cross-linking.

As set forth in the Examples, oxalate decarboxylase crystallizes under anumber of conditions, including a wide pH range (e.g.. pH 3.5 to 8.0). Aprecipitant such as a polyethylene glycol (such as e.g., PEG 200, PEG400, PEG 600, PEG 1000, PEG 2000, PEG 3000, PEG 8000 [See examples 7 and8]) or an organic cosolvent such as 2-methyl-2,4-pentanediol (MPD) isincluded in some embodiments as described. Common salts that may be usedinclude sodium chloride, potassium chloride, ammonia sulfate, zincacetate, etc.

Oxalate decarboxylase may be at a concentration of, e.g., at least 5,10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 mg/ml. ormore in a crystallization broth. The efficiency or yield of acrystallization reaction is at least 50%, 60%, 70%, 80%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. In one embodiment, crystalsof oxalate decarboxylase are grown or produced by a batch process bymixing a solution of oxalate decarboxylase with an appropriate buffer.In certain embodiments, the buffer is 100 mM Tris-HCl buffer, pH 8.0 and100 mM NaCl with 2 mM cysteine-HCl.

Crystallization from cells or cell extract. Crystals may be prepareddirectly from cells or crude cell extracts. In one embodiment, bacteriacells expressing oxalate decarboxylase are harvested. Cells areresuspended with or without DNase and homogenized. A salt solution isadded to the cell lysis to reach a salt concentration of about 0.3 M.0.4 M, 0.5 M, 0.6 M or more. The salt added can be a sodium salt, apotassium salt, a calcium salt, or other salts. Proteins may beoptionally extracted from the cell mixture by removing cell debris. Inone embodiment, homogenized cell mixture is centrifuged, leavingproteins in the supernatant solution. Crystals are generated by reducingsalt concentration of the cell mixture or protein solution. In oneembodiment, salt is removed through dialysis to maintain proteinconcentration. To increase crystal yield, protein solution may beconcentrated before salt concentration of the solution is reduced.Crystals may be generated at a solution with a pH of about 6, 7, or 8.

Crystals may be prepared from a protein precipitate or pellet. In oneembodiment, cells expressing desired proteins are harvested and oxalatedecarboxylase protein is collected in a precipitate or pellet. Pellet orprecipitate containing oxalate decarboxylase protein is solubilized in asalt solution. Crystals are formed by reducing salt concentration in theprotein solution. For increased crystal yields, the salt concentrationin the solubilized protein solution is at least about 0.3 M, 0.4 M, 0.5M or more before it is reduced to produce crystals.

Crystals may also be prepared from a protein solution. In oneembodiment, an oxalate decarboxylase protein solution is concentrated ina salt solution, and crystals are formed when the salt concentration inthe solution is reduced. For increased crystal yields, the saltconcentration is at least about 0.3 M, 0.4 M. 0.5 M or more before it isreduced to produce crystals.

Stabilized crystals. Once oxalate decarboxylase crystals have been grownin a suitable medium they can be optionally stabilized, such as bycross-linking. Cross-linking results in stabilization of the crystallattice by introducing covalent links between the constituent proteinmolecules of the crystal. This makes possible transfer of the proteininto an alternate environment that might otherwise be incompatible withthe existence of the crystal lattice or even with the existence ofintact protein. Oxalate decarboxylase crystals may be cross-linkedthrough, e.g., lysine groups, thiol (sulthydryl) groups, andcarbohydrate moieties. Cross-linked crystals are also referred to hereinas “OXDC-CLEC,” “CLEC-OXDC,” or “CLEC.”

A cross-linked crystal may alter the enzymatic stability (e.g., pH,temperature, mechanical and/or chemical stability), the pH profile ofOXDC activity, the solubility, the uniformity of crystal size or volume,the rate of release of enzyme from the crystal, and/or the pore size andshape between individual enzyme molecules in the underlying crystallattice.

Advantageously, cross-linking, or stabilizing according to the presentinvention carried out in such a way that the crystals comprise an OXDCthat shows at least 60%, 80%, 100%, 150%, 200%, 250%, 300% or more ofthe activity as compared to soluble OXDC. Stability may be increased byat least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%or more as compared to soluble OXDC. Stability can be measured underconditions of storage, such as pH stability, temperature stability,stability against gut proteases, dissolution stability, and as in vivobiological stability, for example.

In some embodiments, cross-linking slows the dissolution of the OXDCpolypeptides in the crystal into solution, effectively immobilizing theprotein, molecules into microcrystalline particles. Upon exposure to atrigger in the environment surrounding the cross-linked proteincrystals, such as under conditions of use rather than storage, theprotein molecules slowly dissolve, releasing active OXDC polypeptideand/or increasing OXDC activity. The rate of dissolution is controlled,for example, by one or more of the following factors: the degree ofcross-linking, the length of time of exposure of protein crystals to thecross-linking agent, the rate of addition of cross-linking agent to theprotein crystals, the nature of the cross-linker, the chain length ofthe cross-linker, pH, temperature, presence of sulfahydryl reagents likecysteine, glutathione, the surface area of the cross-linked proteincrystals, the size of the cross-linked protein crystals, and the shapeof the cross-linked protein crystals.

Cross-linking can be achieved using one or a combination of a widevariety of cross-linking agents, including a multifunctional agent, atthe same time (in parallel) or in sequence. Upon exposure to a triggerin the surrounding environment, or over a given period of time, thecross-links between protein crystals cross-linked with suchmultifunctional cross-linking agents lessen or weaken, leading toprotein dissolution or release of activity. Alternatively, thecross-links may break at the point of attachment, leading to proteindissolution or release of activity. See U.S. Pat. Nos. 5,976,529 and6,140,475.

In some embodiments, the cross-linking agent is a multifunctionalcross-linking agent having at least 2, 3, 4, 5, or more active moieties.In various embodiments, the agent may be chosen from glutaraldehyde,succinaldehyde, octanedialdehyde, glyoxal,dithiobis(succinimidylpropionate), 3,3′dithiobis(sulfosuccinimidylpropionate), dimethyl3,3′-dithiobispropionimidate-HCl,N-succinimidyl-3-(2-pyridyldithio)propionate, hexamethylenediamine,diaminooctane, ethylenediamine, succinc anhydride, phenylglutaricanhydride, salicylaldehyde, acetimidate, formalin, acrolein, succinicsemialdehyde, butyraldehyde, dodecylaldehyde, glyceraldehyde, andtrans-oct-2-enal.

Additional multifunctional cross-linking agents include halo triazines,e.g., cyanuric chloride; halo-pyrimidines, e.g., 2,4,6-trichloro/bromopyrimidine; anhydrides or halides of aliphatic or aromatic mono- ordi-carboxylic acids, e.g., maleic anhydride, (meth)acryloyl chloride,chloroacetyl chloride; N-methylol compounds, e.g., N-methylolchloro-acetamide; di-isocyanates or di-isothiocyanates, e.g.,phenylene-1,4-di-isocyanate and aziridines. Other cross-linking agentsinclude epoxides, such as, for example, di-epoxides, tri-epoxides andtetra-epoxides. In one embodiment, the cross-linking agent isglutaraldehyde, a bifunctional agent, and glutaraldehyde is used aloneor in sequence with an epoxide. Other cross-linking reagents (see, forexample, the 1996 catalog of the Pierce Chemical Company) may also beused, at the same time (in parallel) or in sequence with reversiblecross-linking agents, such as those described below.

According to an alternate embodiment of this invention, cross-linkingmay be carried out using reversible cross-linking agents, in parallel orin sequence. The resulting cross-linked protein crystals arecharacterized by a reactive multi-functional linker, into which atrigger is incorporated as a separate group. The reactive functionalityis involved in linking together reactive amino acid side chains in aprotein and the trigger consists of a bond that can be broken byaltering one or more conditions in the surrounding environment (e.g.,pH, presence of reducing agent, temperature, or thermodynamic wateractivity).

The cross-linking agent may be homofunctional or heterofunctional. Thereactive functionality (or moiety) may, e.g., be chosen from one of thefollowing functional groups (where R, R′, R″ and R′″ may be alkyl, arylor hydrogen groups):

I. Reactive acyl donors, such as, e.g.: carboxylate esters RCOOR′,amides RCONHR′, Acyl azides RCON₃, carbodiimides R—N═C═N—R′, Nhydroxyimide esters, RCO—O—NR′, imidoesters R—C═NH2′ (OR′), anhydridesRCO—O—COR′, carbonates RO—CO—O—R′, urethanes RNHCONHR′, acid halidesRCOHal (where HaI=a halogen), acyl hydrazides RCONNR′R″, and Oacylisources RCO—O—C═NR′(—NR″R′″)

II. Reactive carbonyl groups, such as, e.g.: aldehydes RCHO and ketonesRCOR′, acetals RCO(H₂)R′, and ketals RR′CO2 R′R″ (Reactive carbonylcontaining functional groups known to those well skilled in the art ofprotein immobilization and cross-linking are described in the literature(Pierce Catalog and Handbook, Pierce Chemical Company, Rockford, Ill.(1994); S. S. Wong, Chemistry of Protein Conjugation and Cross-linking,CRC Press, Boca Raton, Fla. (1991));

III. Alkyl or aryl donors, such as, e.g.: alkyl or aryl halides R-Hal,azides R—N, sulfate esters RSO₃ R′, phosphate esters RPO(OR′₃),alkyloxonium salts R₃ O+, sulfonium R₃S+, nitrate esters RONO₂, Michaelacceptors RCR′═CR′″COR″, aryl fluorides ArF, isonitriles RN+=C—,haloamines R₂ N-Hal, alkenes, and alkynes;

IV. Sulfur containing groups, such as, e.g.: disulfides RSSR′,sulfhydryls RSH, and epoxides R₂ C_^(O)CR′₂; and

V. Salts, such as, e.g.: alkyl or aryl ammonium salts R₄ N+, carboxylateRCOO—, sulfate ROSO₃—, phosphate ROPO₃″, and amines R₃ N.

Reversible cross-linking agents, for example, comprise a trigger. Atrigger includes an alkyl, aryl, or other chain with activating groupthat can react with the protein to be cross-linked. Those reactivegroups can be any variety of groups such as those susceptible tonuclcophilic, free radical or electrophilic displacement includinghalides, aldehydes, carbonates, urethanes, xanthanes, and epoxides amongothers. For example, reactive groups may be labile to acid, base,fluoride, enzyme, reduction, oxidation, thiol, metal, photolysis,radical, or heat.

Additional examples of reversible cross-linking agents are described inT. W. Green, Protective Groups in Organic Synthesis, John Wiley & Sons(Eds.) (1981). Any variety of strategies used for reversible protectinggroups can be, incorporated into a cross-linker suitable for producingcross-linked protein crystals capable of reversible, controlledsolubilization. Various approaches are listed, in Waldmann's review ofthis subject, in Angewante Chemie Inl. Ed. Engl., 35:2056 (1996).

Other types of reversible cross-linking agents are disulfidebond-containing cross-linkers. The trigger breaking cross-links formedby such cross-linking agents is the addition of reducing agent, such ascysteine, to the environment of the cross-linked protein crystals.Exemplary disulfide cross-linking agents are described in the PierceCatalog and Handbook (1994-1995). Examples of such cross-linkers andmethods are disclosed in U.S. Pat. No. 6,541,606, relevant portions ofwhich are incorporated by reference.

In addition, cross-linking agents which cross-link between carbohydratemoieties or between a carbohydrate, moiety and an amino acid may also beused.

The concentration of the cross-linking agent may be from about 0.01% to20%, about 0.02% to 10%, or about 0.05% to 5% w/v in solution.Typically, the crosslinking agent is about 0.5% or about 1% w/v. Forexample, the concentration of the cross-linking agent may be, e.g.,about 0.0.1%, 0.02%, 0.05%, 0.075%, 0.1%, 0.2%, 0.3%, 0.5%, 1%, 2%, 3%,3.5%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, or 20% w/v in solution [seeTable 2 in the examples]. It may be necessary to exchange buffers priorto cross-linking, Crystals, including CLECs, may be optionallylyophilized or otherwise formulated.

The crystals, including the cross-linked crystals described herein areuseful in the methods of treatment and methods to reduce oxalate levelsdescribed herein. The OXDC crystals are also useful in methods relatingto industrial processes (e.g., synthesis, processing, bioremediation,disinfection, sterilization), and methods to treat plants, such as plantfungal infections, for example as reviewed in, e.g., Svedruzic el al.,Arch. Biochem. Biophys. 433:176-192 (2005). Such non-therapeuticapplications for soluble or amorphous OXDC are described, for example,in U.S. Pat. Nos. 5,866,778; 6,218,134; 6,229,065; 6,235,530; and6,503,507. The crystals described herein can be applied to these uses,based on one or more properties of the stabilized OXDC crystalsdescribed above, such as increased stability of the oxalatedecarboxylase enzyme.

Drying of Crystals of Oxalate Decarboxylase. Crystals of oxalatedecarboxylase are dried by removal of water, organic solvent or liquidpolymer by means including drying with N₂, air or inert gases, vacuumoven drying, lyophilization, washing with a volatile organic solventfollowed by evaporation of the solvent, evaporation in a fume hood, traydrying, fluid bed drying, spray drying, vacuum drying, or roller drying.Typically, drying is achieved when the crystals become a free flowingpowder. Drying may be carried out by passing a stream of gas over wetcrystals. The gas may be selected from the group consisting of:nitrogen, argon, helium, carbon dioxide, air or combinations thereof.

In principle, dried crystals can be prepared by lyophilization. However,this technique involves rapid cooling of the material and can be appliedonly to freeze stable products. In one embodiment, the aqueous solutioncontaining a crystalline oxalate decarboxylase is first frozen tobetween −40 and −50° C., followed by removal of the under vacuum.

Production of crystals of oxalate decarboxylase, or formulations orcompositions comprising such crystals. In one aspect, crystals ofoxalate decarboxylase, or formulations or compositions comprising suchcrystals are disclosed. Such compositions can be prepared according tothe following process:

First, the oxalate decarboxylase is crystallized. Next, excipients oringredients selected from sugars, sugar alcohols, viscosity increasingagents, wetting or solubilizing agents, buffer salts, emulsifyingagents, antimicrobial agents, antioxidants, and coating agents are addeddirectly to the mother liquor. Alternatively, the mother liquor isremoved, after which the crystals are suspended in an excipient solutionfor a minimum of 1 hour to a maximum of 24 hours. The excipientconcentration is typically between about 0.01 and about 10% (w/w). Theingredient concentration is between about 0.01 and about 90% (w/w). Thecrystal concentration is between about 0.01 and about 99% (w/w).

The mother liquor is then removed from the crystal slurry either byfiltration or by centrifugation. Subsequently, the crystals are washedoptionally with solutions of about 50 to 100% (w/w) of one or moreorganic solvents such as, for example, ethanol, methanol, isopropanol orethyl acetate, either at room temperature or at temperatures betweenabout −20° C. to about 25° C.

The crystals are then dried either by passing a stream of nitrogen, air,or inert gas over them. Alternatively; the crystals are dried by airdrying, spray drying, lyophilization or vacuum drying. The drying iscarried out for a minimum of about 1 hour to a maximum of about 72 hoursafter washing, until the moisture content of the final product is belowabout 10% by weight, most preferably below about 5% by weight. Finally,micromizing (reducing the size) of the crystals can be performed ifnecessary.

According to one embodiment of this invention, when preparing crystalsof oxalate decarboxylase, or formulations or compositions comprisingsuch crystals, enhancers, such as surfactants, are not added duringcrystallization. Excipients or ingredients are added to the motherliquor after crystallization, at a concentration of between about 1 andabout 10% (w/w), alternatively at a concentration of between about 0.1and about 25% (w/w), alternatively at a concentration of between about0.1 and about 50% (w/W). The excipient or ingredient is incubated withthe crystals in the mother liquor for about 0.1 to about 3 hrs,alternatively the incubation is carried out for about 0.1 to about 12hrs, alternatively the incubation is carried out for about 0.1 to about24 hrs.

In another embodiment of this invention, the ingredient or excipient isdissolved in a solution other than the mother liquor, and the crystalsare removed from the mother liquor and suspended in the excipient oringredient solution. The ingredient or excipient concentrations and theincubation times are the same as those described above.

Another advantage of the present invention is that crystals of oxalatedecarboxylase, or formulations thereof, that are encapsulated withinpolymeric carriers to form compositions comprising microspheres can bedried by lyophilization. Lyophilization, or freeze-drying allows waterto be separated from the composition. The oxalate decarboxylase crystalcomposition is first frozen and then placed in a high vacuum. In avacuum, the crystalline water sublimes, leaving the oxalatedecarboxylase crystal composition behind, containing only the tightlybound water. Such processing further stabilizes the composition andallows for easier storage and transportation at typically encounteredambient temperatures.

Spray drying allows water to be separated from the crystal preparation.It is highly suited for the continuous production of dry solids ineither powder, granulate or agglomerate form from liquid feedstocks assolutions, emulsions, and pumpable suspensions. Spray drying involvesthe atomization of a liquid feedstock into a spray of droplets andcontacting the droplets with hot air in a drying chamber. The sprays areproduced by either rotary tea (wheel) or nozzle atomizers. Evaporationof moisture from the droplets and formation of dry particles proceedunder controlled temperature and airflow conditions. Relatively hightemperatures are needed for spray drying operations. However, heatdamage to products is generally only slight, because of an evaporativecooling effect during the critical drying period and because thesubsequent time of exposure to high temperatures of the dry material maybe very short. Powder is discharged continuously from the dryingchamber. Operating conditions and dryer design are selected according tothe drying characteristics of the product and the powder specification.Spray drying is an ideal process where the end product must comply withprecise quality standards regarding particle size distribution, residualmoisture content, bulk density and particle shape.

Compositions. OXDC crystals, including cross-linked crystals areprovided as a composition, such as a pharmaceutical composition (see,e.g., U.S. Pat. No. 6,541,606, describing formulations and compositionsof protein crystals). Pharmaceutical compositions comprising OXDCcrystals include the OXDC crystal with one or more ingredients orexcipients, including, but not limited to sugars and biocompatiblepolymers. Examples of excipients are described in Handbook ofPharmaceutical Excipients, published jointly by the AmericanPharmaceutical Association and the Pharmaceutical Society of GreatBritain, and further examples are set forth below.

The OXDC enzyme may be administered as a crystal in a composition as anyof a variety of physiologically acceptable salt forms, and/or with anacceptable pharmaceutical carrier and/or additive as part of apharmaceutical composition. Physiologically acceptable salt forms andstandard pharmaceutical formulation techniques and excipients are wellknown to persons skilled in the art (see, e.g., Physician's DeskReference (PDR) 2003, 57th ed.. Medical Economics Company, 2002; anRemington: The Science and Practice of Pharmacy, eds. Gennado et al.20th ed, Lippincott, Williams & Wilkins, 2000). For the purposes of thisapplication, “formulations” include “crystal formulations.”

Oxalate decarboxylase useful in the methods of this invention may becombined with an excipient. According to this invention, an “excipient”acts as a filler or a combination of fillers used in pharmaceuticalcompositions. Exemplary ingredients and excipients for use in thecompositions are set forth as follows.

Biocompatiblc polymers. Biocompatible polymers are polymers that arenon-antigenic (when not used as an adjuvant), non-carcinogenic,non-toxic and which are not otherwise inherently incompatible withliving organisms may be used in the OXDC crystal compositions describedherein. Examples include: poly (acrylic acid), poly (cyanoacrylates),poly (amino acids), poly (anhydrides), poly (depsipeptide), poly(esters) such as poly (lactic acid) or PLA, poly (lactic-co-glycolicacid) or PLGA, poly (β-hydroxybutryate), poly (caprolactone) and poly(dioxanone); poly (ethylene glycol), poly((hydroxypropyl)methacrylamide, poly [(organo)phosphazene], poly (orthoesters), poly (vinyl alcohol), poly (vinylpyrrolidone), maleicanhydride-alkyl vinyl ether copolymers, pluronic polyols, albumin,alginate, cellulose and cellulose derivatives, collagen, fibrin,gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans, sulfatedpolysaccharides, blends and copolymers thereof.

Biodegradable polymers, i.e., polymers that degrade by hydrolysis orsolubilization may be included in OXDC crystal compositions. Degradationcan be heterogenous (occurring primarily at the particle surface), orhomogenous (degrading evenly throughout the polymer matrix).

Ingredients such as one or more excipients or pharmaceutical ingredientsor excipients may be included in OXDC crystal compositions. Aningredient may be an inert or active ingredient.

Methods of Treating Oxalate-Associated Disorders with OXDC Crystals. Themethods of the invention comprise administering an oxalatedecarboxylase, e.g., crystals of OXDC or cross-linked forms thereof, toa mammalian subject to treat, prevent, or reduce the risk of occurrenceof a condition associated with elevated levels of oxalate. The elevatedlevels of oxalate may be detected, e.g., in a biological sample from thesubject, such as a body fluid, including urine, blood, serum, or plasma.In certain embodiments, urinary oxalate levels are detected. Thecrystals and/or the compositions comprising crystals may be administeredin the methods described herein.

In some embodiments, methods for treading hyperoxaluria in individualswith primary hyperoxaluria, enteric hyperoxaluria, hyperoxaluria causedby surgical intervention, idiopathic hyperosaluria, oxalosis areprovided. In other instances, elevated oxalate-related disorders of thekidneys, bone, liver gastrointestinal tract and pancreas are amenable totreatment with the methods disclosed herein. Further disorders ordiseases treated by the methods provided herein include, but are notlimited to ethylene glycol (oxalate) poisoning, idiopathic urinary stonedisease, renal failure (including progressive, chronic, or end-stagerenal failure), steatorrhoea, malabsorption, ileal disease, vulvodynia,cardiac conductance disorders, inflammatory bowel disease, cysticfibrosis, exocrine pancreatic insufficiency, Crohn's disease, ulcerativecolitis, nephrocalcinosis, osteoporosis, urolithiasis, andnephrolithiasis. Such conditions and disorders may optionally be acuteor chronic.

The methods of the invention may reduce oxalate levels in a subject byat least 10%, 20%, 30%, 40%, 50%, 60% 70%, 80% 90%, 95%, or more ascompared to levels in an untreated or control subject, to someembodiments, reduction is measured by comparing the oxalate level in asubject before and after administration of OXDC. In some embodiments,the invention provides a method of treating or ameliorating anoxalate-associated condition or disorder, to allow one or more symptomsof the condition or disorder to improve by at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95% or more. In certain embodiments the methodsreduce levels of endogenous oxalate and/or adsorption of dietaryoxalate.

In some embodiments, methods for treating individuals having a genotypeassociated with high oxalate levels are provided, such as individualshomozygous or heterozygous for a mutation that reduces activity of,e.g., alanine:glyoxalate aminotransferase, glyoxylatereductase/hydroxypyruvate reductase, hepatic glycolate oxidase, anotherenzyme involved in oxalate metabolism or associated with hyperoxaluria.In other embodiments, methods for treating individuals having reduced orlacking Oxalobacter formigenes enteric colonization are provided.

The disclosed methods include, administering therapeutically effectiveamounts of oxalate decarboxylase to a mammalian subject at risk for,susceptible to, or afflicted with a condition associated with elevatedlevels of oxalate. The populations treated by the methods of theinvention include, but are not limited to, subjects suffering from, orat risk for developing an oxalate-associated disorder such as, e.g.,primary hyperoxaluria or enteric hyperoxaluria.

Subjects treated according to the methods of the invention include butare not limited to mammals, including humans, non human primates,primates, baboons, chimpanzees, monkeys, rodents (e.g., mice, rats),rabbits, cats, dogs, horses, cows, sheep, goats, pigs, etc.

Indications, Symptoms, and Disease Indicators. Many methods areavailable to assess development or profession of an oxalate-associateddisorder or a condition associated with elevated oxalate levels. Suchdisorders include, but are not limited to, any condition, disease, ordisorder as defined above. Development or progression of anoxalate-associated disorder may be assessed by measurement of urinaryoxalate, plasma oxalate, measurement of kidney or liver function, ordetection of calcium oxalate deposits, for example.

A condition, disease, or disorder may be identified by detecting ormeasuring oxalate concentrations, for example, in a urine sample orother biological sample or fluid. An early symptom of hyperoxaluria istypically kidney stones, which may be associated with severe or suddenabdominal or flank pain, blood in the urine, frequent urges to urinate,pain when urinating, or fever and chills. Kidney stones may besymptomatic or asymptomatic, and may be visualized, for example byimaging the abdomen by x-ray, ultrasound, or computerized tomography(CT) scan. If hyperoxaluria is not controlled, the kidneys are damagedand kidney function is impaired. Kidneys may even fail. Kidney failure(and poor kidney function) may be identified by a decrease in, orlacking urine output (glomerular filtration rate), general ill, feeling,tiredness, and marked fatigue, nausea, vomiting, anemia, anchor failureto develop and grow normally in young children. Calcium oxalate depositsin other tissues and organs may also be detected by methods includingdirect visualization (e.g. in the eyes), x-ray, ultrasound, CT,echocardiogram, or biopsy (e.g., bone, liver, or kidney).

Kidney and liver function, as well as oxalate concentrations, may alsobe assessed using art-recognized direct and indirect assays. Thechemical content or urine, blood or other biological sample may also betested by well known techniques. For example, oxalate, glycolate, andglycerate levels may be measured. Assays for liver and kidney functionare well known, such as, for example, the analysis of liver tissue forenzyme deficiencies and the analysis of kidney tissue for oxalate,deposits. Samples may also be tested for DNA changes known to causeprimary hyperoxaluria.

Other indications for treatment include, but are not limited to, thepresence of one or more risk factors, including those discussedpreviously and in the following sections. A subject at risk fordeveloping or susceptible to a condition, disease, or disorder or asubject who may be particularly receptive to treatment with oxalatedecarboxylase may be identified by ascertaining the presence or absenceof one or more such risk factors, diagnostic, or prognostic indicators.Similarly, an individual at risk for developing an oxalate-relateddisorder may be identified by analysts of one or more genetic orphenotypic markers.

The methods disclosed are useful in subjects with urinary oxalate levelsof at least 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150,160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290,300, ,310, 320, 330, 340, 350, 360, 370, 380, 390, or 400 mg of oxalateper 24 hour period, or more. In certain embodiments, the oxalate levelis associated with one or more symptoms or pathologies. Oxalate levelsmay be measured in, a biological sample, such as a body fluid includingblood, serum, plasma, or urine. Optionally, oxalate is normalized to astandard protein or substance, such as creatinine in urine. In someembodiments, the claimed methods include administration of oxalatedecarboxylase to reduce circulating oxalate levels in a subject toundetectable levels, or to less than 1%, 2%, 5%, 10%, 20%, 30%, 40%,50%, 60%, 70%, or 80% of the subject's oxalate levels prior totreatment, within 1, 3, 5, 7, 9, 12, or 15 days.

Hyperoxaluria in humans can be characterized urinary oxalate excretionof greater than 40 mg (approximately 440 μmol) or 30 mg per day.Exemplary clinical cutoff levels are 43 mg/day (approximately 475 μmol)for men and 32 mg/day (approximately 350 μmol) for women, for example.Hyperoxaluria can also be defined as urinary oxalate excretion greaterthan 30 mg per day per gram of urinary creatinine. Persons with mildhyperoxaluria may excrete at least 30-60 (342-684 μmol) or 40-60(456-684 μmol) mg of oxalate per day. Persons with enteric hyperoxaluriamay excrete at least 80 mg of urinary oxalate per day (912 μmol), andpersons with primary hyperoxaluria may excrete at least 200 mg per day(2280 μmol), for example. Borowski A. E., Langman C B. Hyperoxaluria andOxalosis: Current Therapy and Future directions. Exp Opinion Phrama(2006, in press).

Administration of OXPC Crystals and Compositions Thereof

Administration of oxalate decarboxylase in accordance with the methodsof the invention is not limited to any particular delivery system andincludes administration via the upper gastointestinal tract, e.g., themouth (for example in capsules, suspension, tablets, or with food), orthe stomach, or upper intestine (for example by tube or injection) toreduce oxalate levels in an individual. In certain cases, the OXDC isadministered to reduce endogenous oxalate levels and/or concentrations.OXDC may also be provided by an extracorporeal device, such as adialysis apparatus, a catheter, or a structure or device that contacts abiological sample from an individual.

Administration to an individual may occur in a single dose or in repeatadministrations, and in any of a variety of physiologically acceptableforms, and/or with an acceptable pharmaceutical carrier and/or additiveas part of a pharmaceutical composition (described earlier). In thedisclosed methods, oxalate decarboxylase may be administered alone,concurrently or consecutively over overlapping or nonoverlappingintervals with one or more additional biologically active agents, suchas, e.g., pyridoxine (vitamin B-6), orthophosphate, magnesium,glycosaminoglycans, calcium, iron, aluminum, magnesium, potassiumcitrate, cholestyramine, organic marine hydrocolloid, plant juice, suchas, e.g., banana stem juice or beet juice, or L-cysteine. Biologicallyactive agents that reduce oxalate levels or that increase the activityor availability of OXDC are provided. In sequential administration, theoxalate decarboxylase and the additional agent or agents may beadministered in any order. In some embodiments, the length of anoverlapping interval may be more than 2, 4, 6, 12, 24, or 48 weeks ormore.

The oxalate decarboxylase may be administered as the sole activecompound or in combination with another active compound or composition.Unless otherwise indicated, the oxalate decarboxylase is administered asa dose of approximately from 10 μg/kg to 25 mg/kg or 100 mg/kg,depending on the severity of the symptoms and the progression of thedisease. The appropriate therapeutically effective dose of OXDC isselected by a treating clinician and would range approximately from 10μg/kg to 20 mg/kg, from 10 μg/ka to 10 mg/kg, from 10 μg/kg to 1 mg/kg,from 10 μg/kg to 100 μg/kg, from 100 μg/kg to 1 mg/kg, from 100 μg/kg to10 mg/kg, from 500 μg/kg to 5 mg/kg, from 500 μg/kg to 20 mg/kg, from 1mg/kg to 5 mg/kg, from 1 mg/kg to 25 mg/kg, from 5 mg/kg to 100 mg/kg,from 5 mg/kg to 50 mg/kg, from 5 mg/kg to 25 mg/kg, and from 10 mg/kg to25 mg/kg. Additionally, specific dosages indicated in the Examples or inthe Physician's Desk Reference (PDR) 2003, 57th ed,, Medical EconomicsCompany, 2002, may be used.

The oxalate deearboxylase crystal of the present invention may beadministered through an extracorporeal device or catheter, such as fordelivery of oxalate decarboxylase to a patient. Catheters, for example,urinary catheters, may be coated with Compositions containing oxalatedecarboxylase crystals.

The following Qxamples provide illustrative embodiments of theinvention. One of ordinary skill in the art will recognize the numerousmodifications and variations that may be performed without altering thespirit or scope of the present invention. Such modifications andvariations are encompassed within the scope of the invention. TheExamples do not in any way limit the invention.

EXAMPLES Example 1. Fermentation and Purification of OxalateDecarboxylase

Oxalate decarboxylase (OXDC) from Bacillus subtilis (B. subtilis) is a261 kDa homohexameric protein that consists of six identical monomers.Each monomer contains 385 amino acids, with a calculated molecularweight of ˜43-44 kDa and an isoelectric point of 5.2. The OXDC gene,formerly known as yvrK, was PCR amplified using B. subtilis genomic DNAas template.

The amplified OXDC gene was first cloned into the pCRII vector(Invitrogen, Carlsbad, Calif.) and then subcloned and expressed frompET-11a expression vector using E. coli BL21 (DE3) pLysS cells. Sincegene expression in the pET-11a vectors is under control of the T7promoter, OXDC expression was regulated by induction with IPTG(isopropyl-beta-D-thiogalactopyranoside).

Fermentation was used to achieve high expression levels of recombinantOXDC in E. coli. Expression was performed in 800 L (liter) fermentationmedia containing casein hydrolysate (USB Corporation, Cleveland. Ohio)or soy peptone, yeast extract (USB Corporation), NaCl (FisherScientific), PPG 2000 anti-foam (PPG), KOH (Mallinckrodt Baker, Inc.,Phillipsburg, N.J.), and ampicillin (USB Corporation). Since OXDC is amanganese-dependent enzyme, 5 mM MnCl₂.4H₂O (Mallinckrodt Baker, Inc.)was included in the fermentation medium. Expression of OXDC was inducedby addition of 0.4 mM IPTG (Lab Scientific). The cells expressing OXDCwere grown either in shake flasks or in a fermenter. By this method,OXDC was expressed mainly in pellet preparations,

A glycerol stock of BL21 cells transformed with pET11a-OXDC was used forfermentation. For 800 L fermentation, a pre-seed culture was preparedwith 2×250 ml flasks, each containing 50 ml of medium (LB plus 100 μg/mlof ampicillin). 2×0.5 ml of inoculum was used. The cultures wereincubated at 35° C., 250 rpm for 6 hours. This culture was thentransferred to 6×2 L flasks each containing 1 L of media (LB plus 100μg/ml of ampicillin). In each flask, 10 ml of starter culture was added.These cultures were incubated at 35° C., 250 rpm for 12 hours. 6 L ofthis culture was transferred to the 800 L fermenter containing theappropriate media as described above. The culture was grown at 37° C.,pH 7.0, shaking at 100 rpm with dissolved oxygen around 30-40 until theOD₆₀₀ reached about 0.3 (this process took approximately 2-3 hours).Expression of OXDC was induced with 0.4 mM of IPTG plus 5 mM MnCl₂.4H₂O.The culture was induced for 4 hours at 37° C., 100 rpm. The cells werethen harvested and frozen for later use. The OD₆₀₀ at the time ofharvesting was about 5.5-8.0.

Cells were resuspended in a ratio of 1 kg cell paste per 4 L buffercontaining 50 mM Tris pH 8, 100 mM NaCl, and in the presence or absenceof 15-25 U/ml DNase. Cell suspension was blended (50-60 rpm) overnightat 4° C. The cell suspension was passed through a pre-cooled homogenizerthree times on ice. The efficiency of cell lysis was checked under themicroscope and unbroken cell suspension was used as a control. Cellswere centrifuged at 4,000 rpm for 40 min in 1 L bottles at 4° C.Supernatant and pellet were saved for characterization by SDS-PAGE. Theexpression of OXDC was analyzed by SDS-PAGE. The majority of the OXDCwas found in the pellet, which included inclusion bodies and otherprecipitates. The pellet was harvested by centrifugation at 4000 rpm for40 min and was used immediately or frozen at −70° C. for later use.

From an 800 L fermentation reaction, we obtained between 5,000 and 5,500g of cells, which yielded between 2,800 and 3,000 g of wet weightpellets.

Example 2. Crystallization of Oxalate Decarboxylase from SolubilizedPellets with Mild Denaturant Concentration Followed by Anion ExchangeChromatography

OXDC pellets stored frozen at −20° C. were used to prepare crystals ofOXDC.

In this procedure, pellets were solubilized under mild conditions ofdenaturant concentration and pH. The solubilized protein was thenrefolded using an anion exchange matrix column.

The pellets were solubilized in 2 M Urea, 100 mM Tris pH 10.0, 10 mM DTTand 100 mM NaCl (1:10 w/v). The solution was stirred at room temperature(RT) for 2 h, and then the solution was centrifuged at 15 K. for 30 minat 4° C. The supernatant was carefully decanted and saved. The pelletwas carefully weighed and saved separately.

The solubilized pellets in supernatant were added dropwise, at a flowrate of 10 ml/min, to 10 vol. of a solution consisting of 2 M Urea, 100mM Tris pH 8.0, 1 mM DTT, and 1 mM MnCl₂, with constant and gentlestirring. After complete addition of the supernatant the proteinsolution was incubated at RT for 1 h. After incubation, the proteinsolution was made ready for anion exchange chromatography bycentrifuging at 15 K for 30 min at 4° C. to remove any possibleprecipitation.

An anion exchange chromatography column was prepared by packing Qsepharose matrix in glass column. The column was attached to an FPLC andequilibrated by washing with 10 column volumes (CV) of a solution of 0.5M Urea, 100 mM Tris pH 8.0, 1 mM DTT, and 1 mM MnCl₂. The flow-rate wasmaintained at 6 ml/min. Solubilized pellet was loaded onto the column.Protein load was 8-10 mg/ml of matrix. After loading the sample, thecolumn was washed with at least 10 CV of 100 mM Tris pH 8.0, 1 mM DTTand 1 mM MnCl2 at a flow rate of 6 ml/min. This step removed urea fromthe bound protein sample and allowed the protein to refold to its nativeconformation.

The protein was eluted from the column with 1 M NaCl, 100 mM Tris pH8.0, and 1 mM DTT by either a step gradient or by step elution. Theprotein elution was monitored at 280 nm and fractions of 10 ml werecollected. The peak fractions were pooled together and tested bySDS-PAGE and enzyme activity.

The peak fractions containing OXDC were concentrated to 15 mg/ml bystirring cells using a 10,000 MWCO membrane, and dialyzing against 10vol. of 100 mM Tris pH 8.0, 100 mM NaCl, and 1 mM DTT. Two changes ofbuffer were made at one hour intervals and after a third change ofbuffer, the dialysis continued overnight for maximum crystal recovery.Protein crystallized in dialysis bags was recovered by centrifuging thesample at 2K, for 15 min at 4° C. After dialysis, about 70% of therefolded OXDC was crystallized. The crystals were cubic in shape and ofuniform size. The crystals showed an activity of ˜44 units.

Example 3. Crystallization of Oxalate Decarboxylase by SolubilizingPellets Using High Denaturant Concentration Followed by Anion ExchangeChromatography

By this method, pellets were solubilized in 5 M urea, 50 mM Tris pH 8.6,100 mM NaCl, 10 mM DTT (1:5 w/v). The solution was stirred at RT for 2h, and then the solution was centrifuged at 15K for 30 min at 4° C. Thesupernatant was carefully decanted and saved. The pellet was weighed andsaved separately.

Anion exchange chromatography column was prepared by packing Q sepharosematrix in glass column. The column was attached to an FPLC andequilibrated by washing with 3 column volumes (CV) of the 4 M Urea, 100mM Tris pH 8.6 and 10 mM DTT. The column was further washed with 7 CV in100 mM NaCl, 50 mM Tris pH 8, 1 mM MnCl₂, 10 mM DTT and eluted in asingle step with 3 CV of 0.5 M NaCl, 50 mM Tris pH 8.0, 1 mM DTT, 1 mMMnCl₂.

Appropriate fractions were collected and the protein was identified bySDS-PAGE and activity assay. The soluble protein was concentrated in thepresence of high salt (0.5 M NaCl). A total volume of 450 ml was elutedwith 0.5 M salt and concentrated to 45 ml with pellicon filtration into100 mM NaCl, 50 mM Tris pH 8.0, 1 mM DTT. After protein concentration,dilution was performed in order to reduce the salt concentration from0.5 M to 0.1 M NaCl. At this point, crystals of OXDC begun to form. Inthis example, the volume was brought to 210 mL in 100 mM NaCl, 50 mMTris pH 8.0, 1 mM DTT equivalent. Formed crystals were spun andrecovered in 100 mM NaCl, and 50 mM Tris pH 8.0, in the presence orabsence of 1 mM DTT.

Example 4. Crystallization of Oxalate Decarboxylase by SolubilizingPellets Using High pH and Mild Denaturant Concentration Followed byConcentration on Hollow Fiber

Pellets Containing Inclusion Bodies and Other Precipitates (3.93 kg)were solubilized in 9.5 L of 50 mM Tris pH 12, 500 mM NaCl, 2 M urea, 10mM DTT for 2 hours at room temperature. Final volume was 12 L. pH 9.9.The sample was spun at 7000 rpm for 45 minutes and supernatant wasrecovered. The total volume after spinning was 11.1 L, pH 9.9. Thesample was concentrated on hollow fiber to 5 L for an hour and thevolume was then slowly brought up to 20 L with 50 mM Tris pH 8.0. 500 mMNaCl, 2 M urea, 10 mM DTT. This process was done for an hour. The finalconcentration of urea at this point is estimated around 0.5 M urea. Thevolume was concentrated to 6 L in hollow fiber for 2.5 h. The sample wasdiluted to 24 L with 50 mM Iris pH 8.0, 500 mM NaCl, 1 mM DTT, 1 mMMnCl, 200 mM L-Arginine for 30 min. At this point the concentration ofurea was estimated to be 125 mM. Another round of concentration was doneto a final volume of 5 L. The sample was diluted to 18 L with 50 mM TrispH 8.0, 500 mM NaCl, 1 mM DTF, 1 mM MnCl₂ and concentrated again to 6.5L. The pH was at this time 8.1. The sample was spun 45 minutes at 7000rpm and the final pellet was saved for analysis. After centrifugation, adilution step with 50 mM Tris pH 8.0, 1 mM DTT, was performed to obtaincrystals. The dilution was to 30 L using a peristaltic pump, withmixing, at room temperature. The flow rate of dilution was estimated tobe 50 ml/min. This dilution took about 9 hours. Crystals were harvestedby centrifugation and the supernatant was saved for analysis. Thecrystals were washed three times with 50 mM Tris, 100 mM NaCl pH 8 andresuspended in 50 mM Tris, 100 mM NaCl pH 8. The crystals were stored at4° C.

Example 5. Crystallization of Oxalate Decarboxylase from Cell ExtractsUsing High Salt

(1) Crystallization by Solubilizing Protein-Containing Pellets UsingHigh Salt Concentration Followed by Concentration and Dilution

Frozen pellets (465 g) of Example 1 were solubilized in 2.3 L 100 mMTris, 1 mM L-cysteine HCL, 0.5 M NaCl, pH 8.0, for 2 hours at roomtemperature, forming soluble oxalate decarboxylase. The sample was spunat 7000 rpm for 45 minutes and supernatant was recovered. The finalvolume was 2.15 L and the protein concentration measured was 24.14mg/ml. The sample was concentrated to 550 ml by tangential flowfiltration (10 kD Pall) for an hour, and then diluted to 2,750 L with100 mM Tris pH 8.0, 1 mM L-cysteine HCl over 30 minutes at a flow rateof 73 ml/min and stirring for an hour at room temperature. Crystals wereallowed to form overnight in the cold room. Crystals were harvested bycentrifugation, and the supernatant was saved for analysis. The crystalswere washed three times with 100 mM Tris. 100 mM NaCl pH 8 and thenresuspended in 100 mM Tris, 100 mM NaCl pH 8. The crystals were storedat 4° C. Recombinant B. subtilis is OXDC purified from E.coli expressionmedium exhibits a specific activity of about 50-60 U/mg under standardassay conditions (see Example 15).

(2) Crystallization by Solubilizing Pellets Using High SaltConcentration Followed by Concentration and Dialysis

Frozen pellets (510 g) of Example 1 were solubilized in 3 L 100 mM Tris,1 mM L-cysteine HCL, 0.5 M NaCl, pH 8.0, for 2 hours at roomtemperature. The sample was spun at 7000 rpm for 30 minutes andsupernatant was recovered. The sample was concentrated to 500 ml bytangential flow filtration (10 kD Pall) for an hour, and then dialyzedagainst 100 mM Tris pH 8.0 with stirring. Crystals were allowed to formovernight in the cold room. Crystals were harvested by centrifugation,and the supernatant was saved for analysis. The crystals were washedthree times with 100 mM Tris, pH 8.0 and then resuspended in 100 mM TrispH 8.0. The crystals were stored at 4° C. The crystal yield was 60%.

(3) Crystallization by Solubilizing Pellets Using High SaltConcentration Followed by Concentration and Dialysis

Frozen pellets (510 g) of Example 1 were solubilized in 3 L 100 mM Tris,1 mM L-cysteine HCL, 0.5 M NaCl, pH 8.0, for 2 hours at roomtemperature. The sample was spun at 7000 rpm for 30 minutes andsupernatant was recovered. The sample was concentrated to 500 ml bytangential flow filtration 10 kD Pall) for an hour, and then dialyzedagainst 100 mM Tris pH 7.5 with stirring. Crystals were allowed to formovernight in the cold room. Crystals were harvested by centrifugation,and the supernatant was saved for analysis. The crystals were washedthree times with 100 mM Tris, pH 7.5 and then resuspended in 100 mM IrispH 7.5. The crystals were stored at 4° C. The crystal yield was 67%.

(4) Crystallization by Solubilizing Pellets Using High SaltConcentration Followed by Concentration and Dialysis

Frozen pellets (510 g) of Example 1 were solubilized in 3 L 100 mM Tris,1 mM HCL, 0.5 M NaCl, pH 8.0, for 2 hours at room temperature. Thesample was spun at 7000 rpm for 30 minutes and supernatant wasrecovered. The sample was concentrated to 500 ml by tangential flowfiltration (10 kD Pall) for an hour, and then dialyzed against 100 mMTris pH 7.0 with stirring. Crystals were allowed to form overnight inthe cold room. Crystals were harvested by centrifugation, and thesupernatant was saved for analysis. The crystals were washed three timeswith 100 mM Tris, pH 7.0 and then resuspended in 100 mM Tris pH 7.0. Thecrystals were stored at 4° C. The crystal yield was about 80%.

(5) Crystallization by Solubilizing Pellets Using High SaltConcentration Followed by Concentration and Dialysis

Frozen pellets (510 g) of Example 1 were solubilized in 3 L 100 mM Tris,1 mM HCL, 0.5 M NaCl, pH 8.0. for 2 hours at room temperature. Thesample was spun at 7000 rpm for 30 minutes and supernatant wasrecovered. The sample was concentrated to 500 ml by tangential flowfiltration (10 kD Pall) for an hour, and then dialyzed against 100 mM.sodium citrate buffer pH 6.5 with stirring. Crystals were allowed toform overnight in the cold room. Crystals were harvested bycentrifugation, and the supernatant was saved for analysis. The crystalswere washed three times with 100 mM sodium citrate buffer, pH 6.5 andthen resuspended in 100 mM sodium citrate buffer pH 6.5. The crystalswere stored at −4° C. The crystal yield was about 70%,

(6) Crystallization by Solubilizing Pellets Using High SaltConcentration Followed by Concentration and Dialysis

Frozen pellets (510 g) of Example 1 were solubilized in 3 L 100 mM Tris,1 mM L-cysteine HCL, 0.5 M NaCl, pH 8.0, for 2 hours at roomtemperature. The sample was spun at 7000 rpm for 30 minutes andsupernatant was recovered. The sample was concentrated to 500 ml bytangential flow filtration (10 kD Pall) for an hour, and then dialyzedagainst 100 mM sodium citrate butter pH 6.0 with stirring. Crystals wereallowed to form overnight in the cold room. Crystals were harvested bycentrifugation, and the supernatant was saved for analysis. The crystalswere washed three times with 100 mM sodium citrate buffer, pH 6.0 andthen resuspended in 100 mM sodium citrate butter pH 6.0. The crystals were stored at 4° C. The crystal yield was about 60%.

(7) Crystallization of OXDC from Cell Paste After Homogenization andSolubilization

Cells were resuspended in a ratio of 1 kg cell paste per 3 L buffercontaining 100 mM Tris pH 7.5, 500 mM NaCl, 5 mM cysteine, and 1 mMmanganese chloride. Cell suspension was blended (50-60 rpm) overnight at4° C. The cell suspension was passed through a pre-cooled homogenizertwo times on ice. The efficiency of cell lysis was checked under themicroscope and unbroken cell suspension was used as a control. Thesuspension was made up to 10 L and mixed with overhead stirrer for 3 hrsat room temperature. The crude extract was then centrifuged at 7,000 rpmfor 30 min at 4° C. and supernatant was recovered. Supernatant andpellet were saved for characterization by SDS-PAGE. The expression ofOXDC was analyzed by SDS-PAGE. The majority of the OXDC was found in thesupernatant. The final volume was 10 L and the protein concentrationmeasured was 34 mg/ml. The sample was concentrated to 3.5 L bytangential flow filtration (10 kD Pall) for an hour, and then dilutedusing crystallization buffer (100 mM Tris. 100 mM NaCl pH 7.5) withstirring for an hour at room temperature. Crystals were harvested bycentrifugation, and the supernatant was saved for analysis. The crystalswere washed three times with 100 mM Tris, 100 mM NaCl pH 7.5 and thenresuspended in 100 mM Tris and 100 mM NaCl, pH 7.5. The crystals werestored at 4° C.

Example 6. Crystallization of OXDC from Soluble Protein

OXDC was expressed in E. coli in a shaking flask. The cells were lysedby microfluidizer with 25 mM Tris-HCl buffer, pH 8.0, 100 mM NaClcontaining 25 U/ml of DNase I. The cell lysate was incubated at roomtemperature for one hour to allow OXDC crystals to form. The crystalswere spun down and reconstituted in 100 mM Tris, 100 mM NaCl pH 8.

Example 7. Crystallization of OXDC by Vapor Diffusion

Hanging drop crystallization trials were performed using commerciallyavailable sparse matrix crystallization kits: Crystal Screen (HamptonResearch; Aliso Viejo, Calif.), Crystal Screen 2 (Hampton Research),Wizard I (Emerald Biosystems; Bainbridge Island, Wash.), Wizard II(Emerald Biosystems), Cryo I (Emerald Biosystems), and Cryo II (EmeraldBiosystems).

600 μl of reagent was placed in each well. 3 μl of reagent was dispensedonto a glass microscope coverslip and 3 μl of OXDC was dispensed intothe reagent drop with minimal mixing. Up to five more drops were madefrom this 6 μl reagent and OXDC drop. As the drops were minimally mixed,each of the subsequent (smaller) drops had a different and unknown ratioof protein to reagent, thereby increasing the likelihood of obtainingcrystals in a short period of time. The hanging drops were examined forcrystals under a microscope after overnight incubation at roomtemperature. A large number of crystallization conditions were obtained,as shown in Table 1.

TABLE 1 Crystallization conditions for OXDC in hanging drops.^(a)Description Precipitant of Crystals 40% (v/v) Ethanol, 0.1M phosphatecitrate, needles pH 4.20, 5% (w/v) PEG 1000 20% (w/v) PEG-3000, 0.1MHEPES, pH 7.50, rods 0.2M NaCl 20% (w/v) PEG-3000, 0.1M acetate pH 4.5Rods, plates 20% (w/v) PEG 8000, 0.1M phosphate-citrate, plates pH 4.20,0.2M NaCl 10% (w/v) PEG 8000, 0.1M Cacodylate, pH 6.50, rods 0.2MMagnesium acetate 9% (w/v PEG 8000, 0.1M Cacodylate, pH 6.50, needles0.2M Calcium acetate 40% (v/v) PEG 400, 0.1M Na/phosphate, needles pH6.20, 0.2M NaCl 10% (v/v) PEG 8000, 0.1M Na/phosphate, rods pH 6.20,0.2M NaCl 20% (w/v) PEG 2000 MME, 0.1M Tris, pH 7.0 plates ^(a)OXDCconcentration was determined by Bradford assay to be about 1.7 mg/mL.

Example 8. Crystallization of OXDC by Microbatch

Oxalate decarboxylase could be crystallized by the microbatch methodfrom a number of crystallization conditions:

-   -   (i) 10 μl of purified OXDC at a concentration of 23.46 mg/ml was        mixed with 10 μof 16% PEG 8000. Crystallization occurred        immediately in 2-5 seconds. Big crystals formed with some        precipitates.

ii) 10 μl of purified OXDC at a concentration of 23.46 mg/ml was mixedwith 10 μl of 20% PEG 8000. Crystallization occurred immediately in 2-5seconds. Big crystals formed with no precipitates.

(iii) 10 μl of purified OXDC at a concentration of 23.46 mg/ml was mixedwith 10 μl of 24% PRO 8000. Crystallization occurred immediately in 2-5seconds. Smaller cube shaped crystals formed.

(iv) 10 μl of purified OXDC at a concentration of 23.46 mg/ml was mixedwith 10 μl of 28% PRO 8000. Crystallization occurred immediately in 2-5seconds. Very small cube shaped crystals formed. There was noprecipitation.

(v) 8 μl of purified OXDC at a concentration of 23.46 mg/ml was mixedwith 12 μl of 24% PEG 8000. Crystallization occurred immediately in 2-5seconds. Very small cube shaped crystals formed. There was noprecipitation.

(vi) 9 μl of purified OXDC at a concentration of 23.46 mg/ml was mixedwith 11 μl of 24% PEG 8000. Crystallization occurred immediately in 2-5seconds. Small cube shaped crystals formed. There was no precipitation.

(vii) 10 μl of purified oxalate decarboxylase at a concentration of23.46 mg/ml was mixed with 10 μl of 24% PEG 8000. Crystallizationoccurred immediately in 2-5 seconds. Small cube shaped crystals formed.There was no precipitation.

(viii) 11 μl of purified oxalate decarboxylase at a concentration of23.46 mg/ml was mixed with 9 μl of 24% PEG 8000. Crystallizationoccurred immediately in 2-5 seconds. Cube shaped crystals formed. Therewas no precipitation.

(ix) 12 μl of purified OXDC at a concentration of 23.46 mg/ml was mixedwith 8 μl of 24% PEG 8000. Crystallization occurred immediately in 2-5seconds. Cube shaped crystals formed. There was no precipitation.

Example 9. Activities of Soluble OXDC and OXDC Crystals

Soluble OXDC was collected as described in Example 5 after the pelletswere solubilized, spun and the supernatant recovered. OXDC crystals werecollected as described in Example 5 after the crystals were harvestedand washed. Activities of soluble OXDC and OXDC crystals were measuredaccording to Example 15. In one experiment, the activity of soluble OXDCwas 12 Units/mg, and the activity of OXDC crystal was 35 Units/mg.

Soluble OXDC is collected as described in any of Examples 2-5 and OXDCcrystals are harvested as described in any of Examples 2-8. Activitiesof soluble and crystal OXDC are measured according to Example 15.Activities of OXDC crystals can be at least about 100%, 200%, 300%, 400%or 500% of the activities of soluble OXDC.

Example 10. Cross-Linking of Oxalate Decarboxylase Crystals withGlutaraldehyde.

Oxalate decarboxylase crystals prepared according to any of the Examples2-8 were cross-linked using glutaraldehyde. After crystallization, OXDCcrystals were concentrated to 20-30 mg/ml. 0.8 ml of 25% glutaraldchydewas added to 20 ml of crystals to make a solution of 1% glutaraldehyde,and crystals were tumbled for 18 hours at room temperature. Cross-linkedcrystals were washed five times with 100 mM Tris, pH 7.00 andresuspended in 10 mM Tris, pH 7.00.

Specific activities of crystalline OXDC and cross-linked OXDC (referredto as OXDC-CLEC) were compared (six trials) and it was shown thatcross-linked oxalate decarboxylase crystals retain more than 30% to morethan 50% of the original activity of the crystalline protein, in variouspreparations.

To test the effects of varying concentrations of glutaraldehyde onenzyme activity, 1 ml aliquots of OXDC crystals (60 mg/ml) werecrosslinked with different concentrations of glutaraldehyde (from 0.05%to 2%, final concentration) at pH 8.0 at 25° C. for 18 hrs. Thecrosslinking was terminated by separation of the crosslinked crystals bycentrifugation at 2000 rpm in an Eppendorf tube and then resuspension ofthe crosslinked crystals in 1 ml of 100 mM Tris.HCl pH 7.0. Thecross-linked OXDC (OXDC-CLEC) was then washed five times with 100 mMTris HCl buffer, pH 7.5, followed by three washes with 10 mM Tris HClbuffer, pH 7.5 (see results in Table 2. below).

Example 11. pH Controlled Solubility of Crosslinked OxalateDecarboxylase Crystals

Solubility of various crosslinked oxalate decaroboxylase crystals wasexamined following a decrease in pH from 7.5 to 3.0. The cross-linkedcrystals were incubated at 1 mg/ml in 50 mM glycine-HCl (pH 3.0).Aliquots were removed after 5 hour incubation at 37° C. with stirring.Soluble protein concentration was measured at OD₂₈₀ nm after separationof the undissolved crosslinked crystals by centrifugation at 2000 rpmand filtration of the supernatant through 0.22 μm filter. The resultsare described in Table 2 below.

TABLE 2 Crosslinking of OXDC Crystals with Different Percentages ofGlytaraldehyde and pH-Controlled Solubility of OXDC-CLEC Sample %Glutaraldehyde % Protein Leaching OxDc-CLEC-1 0.005 100.0 OxDc -CLEC-20.010 100.0 OxDc -CLEC-3 0.050 2.2 OxDc -CLEC-4 0.075 0.0 OxDc -CLEC-50.100 0.0 OxDc -CLEC-6 0.200 0.0 OxDc -CLEC-7 1.000 0.0

These results indicate that a substantially stable glutaraldehyde OXDCcrosslinked crystal is formed in the presence of at least about 0.05%(final concentration) glutaraldehyde.

Oxalate decarboxylase crystals, prepared as described in Examples 2-8,were crosslinked by addition of glutaraldehyde (Sigma). A 1 ml aliquotof OXDC crystals (30-40 mg/ml) was crosslinked with 1% glutaraldehyde(final concentration) at pH 8.0 at 25° C. for 18 hrs. The crosslinkingwas terminated by separation of the crosslinked crystals bycentrifugation at 2000 rpm in an Eppendorf tube and then resuspension ofthe crosslinked crystals in 1 ml of 100 mM Tris HCl, pH 7.0. TheOXDC-CLEC was then washed five times with 100 mM Tris-HCl buffer, pH7.0, followed by three washes with 10 mM Tris-HCl buffer, pH 7.0. The pHactivity profile of OXDC-CLEC was assayed by measuring the activity ofthe crystals as described in Example 15 using various buffers and pHs:50 mM glycine-HCl buffer at pH 2.0 and 3.0; 50 mM succinate buffer atpHs 4.0, 5.0, and 6.0; and 50 mM Tris buffer at pH 7.0. The activitylevel at each pH was performed twice and the average activity wascalculated. The results, shown in FIG 1, indicate that OXDC-CLEC is mostactive than its soluble counterpart between pH 3.5 and 6.0. Theuncrosslinked crystals showed much higher activity, ranging from about50% to about 200%, 300% or 400% more, than the soluble form of oxalatedecarboxylase at different pH.

Example 13. Oxalate Decarboxylase Therapy in Animal Model for EntericHyperoxaluria

Rat Model for Enteric Hyperoxaluria, Dose Range Study: Male SpragueDawley (SD) rats fed a diet high in oxalate constitute a suitable animalsystem for the study of enteric hyperoxaluria. In this study,administration of 1.1% dietary potassium oxalate resulted in a 5- to10-fold increase in urinary oxalate.

Twenty Sprague Dawley (SD) rats less than 35 days old and weighing100-120 grams were randomly divided into a control group andexperimental groups (five rats per group). Rats were acclimated for 7days to individual metabolic cages (LabProducts, Inc.; Seaford, Del.)prior to treatment. During this period, rats were provided ad libitumwith supplemented acidified water and fed a synthetic diet having 1.1%potassium oxalate and a low (0.5%) concentration of calcium (ResearchDiets TD89222PWD: Harlan Teklad; Madison, Wis.). Rats were maintained onthis diet for the duration of the treatment.

Following the acclimation period, three different doses of recombinantoxalate decarboxylase, formulated as cross-linked crystals with 1%glutaraldehyde (see, e.g., Example 9), were administered to the testrats for 4 consecutive weeks. The crystals were administered orally as afreeze/dried food enzyme mixture (5, 25 and 80 mg of OXDC-CLEC slurry in10 mM Tris HCl pH 7.0, each separately mixed with 15 g food and freezedried; each morning, food containers were re-filled with ˜20 g offood/enzyme mixture). Prior to the treatment, rats were randomly dividedbetween a control group and experimental group based on their basalurinary oxalate.

Analysis of urine samples: 24 hour urinary samples were collected inmetabolic cages over acid (250 μl of 6N hydrochloride acid was mixedwith urine sample collected during 24 h) in order to minimize thespontaneous breakdown of urinary ascorbic acid to oxalate. Samples werestored at −70T until further analysis. Daily diuresis and multiple 24(hour) urine samples were collected for oxalate and creatininemeasurements. Assays for oxalate and creatinine are described in Example15. Urinary excretion of oxalate and creatinine were expressed as μmolof oxalate and creatinine detected in 24 h urine samples. All data wereanalyzed statistically using the Student's t-test.

As shown in FIG. 2, oral administration of OXDC-CLEC to SD rats withchronic hyperoxaluria resulted in a sustained reduction of urinaryoxalate from day 4 of the treatment. Maximal continuous reductionbetween 25-40% was recorded in the highest dose group (80 mg(OXDC-CLEC). The lower doses of 5 mg and 25 mg OXDC-CLEC producedsmaller reduction in urinary oxalate (up to 30 in the 25 mg group and20% in the 5 mg group, respectively). The doses of 25 mg and 80 mgproduced significant is reduction during, all tested days (except day 21for 25 mg group), while the lowest dose of 5 mg had a minimal effectwhich was not significant. This result reveals a dose dependant effectof OXDC-CLEC therapy.

Example 14: Oral Oxalate Decarboxylase therapy in animal model forprimary Hyperoxaluria

Mouse Model For Type 1 Primary Hyperoxaluria: AGT1 knockout mice lackthe liver peroxisomal enzyme alanine:glyoxylate aminotransferase, adeficiency that causes primary hyperoxaluria, Type 1. Chimeric mice werebred to homozygosity in (C57B16 and 129/sv background strains. Allhomozygous Agxt mice showed mild hyperoxaluria 1-2 mmol/L), 5-10 foldurinary oxalate elevation over normal values, as compared to wildtype(0.2 mmol/L). It was also found that 30-50% of males, and 0% of femalesdeveloped mild nephrocalcinosas and calcium oxalate calculi in theurinary tract later in life (4-7 months of age). Interestingly, when themutation was analyzed in a homogeneous (C57B16 strain, hyperoxaluria wasnot associated with development of urinary stones in either sex;underscoring the phenotypic variability typically observed in thisdisease.

A total of 44 male mice (strain ATG1 KO/129sv, developed by Dr, Salido,La Laguna Tenerife, Spain) were used in these experiments. Mice wererandomly divided between a control group and three experimental groups.Mice weighed 20-25 grams and were less than 6 months of age.

The AGT1 KO (129sv) mice were challenged with ethylene glycol (EG) toprovoke severe hyperoxaluria and the formation to calcium oxalatedeposits in the kidney parenchyma. EG is a common alcohol that ismetabolized in the liver to oxalate. Usually, after 2-6 weeks of EGchallenge, AGT1 KO mice in the 129/sv background show signs of impairedkidney function as determined by (i) the variable excretion of oxalatein the urine, (ii) decreased creatinine clearance, and (iii)nephrocalcinosis that ultimately leads to renal failure and death.

Mice were acclimated for 7 days prior to treatment to individualmetabolic cages (Teeniplast USA Inc. Exton, Pa., USA), and were fedstandard breeder diet (17% proteins, 11% fat, 53.5% carbohydrate)containing less than 0.02-0.08% oxalate and approximately 0.5-0.9%calcium. After the acclimation period, mice were divided into fourgroups; three treatment groups were fed oxalate decarboxylase-CLEC mixedwith food, while mice in the matched control group received the samediet without addition of the test article. Drinking water supplementedwith 0.7% EG was provided to all mice ad libitum from the first day oftreatment until the end of the study. After several days of challenge,mice excrete approximately 3-6 mmol/L oxalate in their urine per day,which is approximately 10-20 fold more than wild type (unchallenged)mice.

Administration of OXDC-CLEC Enzyme; Dose Range Study: A total of 44 malemice from strain AGT1 KO/129sv were used in a dose study of OXDC-CLEC.The mice weighed 20-25 grams and were less than 6 months of age. Themice were challenged with EG, and then were randomly divided between acontrol group and an experimental group. The efficacy of three differentdoses of recombinant oxalate decarboxyl formulated as cross-linkedcrystals (1% glutaraldehyde; see Example 9), was monitored over fourconsecutive weeks. The term “OXDC-CLEC as used in this Example refers torecombinant oxalate decarboxylase, formulated as cross-linked crystals(1% glutaraldehyde, as described in Example 9. OXDC-CLEC was orallyadministered as a freeze/dried food enzyme mixture at nominal doses of5, 25 and 80 mg/day. An adequate amount of enzyme slurry in 10 mMTris-HCl buffer (pH 7.0) was mixed with 3.5 g food and freeze dried.Each morning, food containers were re-filled with ˜7 g of thefood/enzyme mixture.

Assessment of the efficacy of OXDC-CLEC: The efficacy of the enzymetherapy was monitored by urinary oxalate reduction, prevention ofcalcium oxalate deposition in kidney parenchyma, and survival. At theend of the study, surviving mice were sacrificed and blood samples takenfor creatinine measurement.

Analysis of urine samples: 24 h urinary samples were collected inmetabolic cages over acid (50 μl of 6 N hydrochloride acid per 3-4 ml ofurine) in order to minimize the spontaneous breakdown of urinaryascorbic acid to oxalate. Urine samples were stored at −20° C. untilfurther analysis. Daily diuresis and multiple 24 h urine samples werecollected and analyzed for oxalate and creatinine levels. Assays foroxalate and creatinine are described in Example 15. Urinary excretion ofoxalate and creatinine was expressed as μmol of oxalate or creatinineexcreted in 24 h urine sample (mL). Data were analyzed statisticallyusing Student's t-test.

Analysis of blood samples: At the end of the study, mice were sacrificedand serum samples were collected. For scrum creatinine measurement, aslightly modified version of the Jaffe reaction method (see, e.g., theCreatinine Microassay Plate Kit from Oxford Medical Research. Inc.;Slot, Scand J. Clin. Lab. Invest. 17:381, 1965; and Heinegard D, Clin.Chim. Acta 43:305, 1973) was used. 80 μl of undiluted serum samples wasmixed with 800 μl of picric alkaline in the cuvettes and incubated for30 minutes at room temperature. Color development was measuredspectrophotometrically at 510 nm; 33.3 μl of 60% acetic acid was thenadded to quench the unspecific reaction. Samples were thoroughly mixedand after 5 minutes incubation at room temperature were read again at510 nm. Final absorbance is present as a difference of two readings.Serial dilutions of 1 mM creatinine solution was used for a standardcurve.

Kidney function was monitored indirectly by measuring creatinineclearance. Creatinine clearance is expressed as excretion rate ofcreatinine (U_(er)×V), where U_(er) represents the concentration ofcreatinine (μmol/L) in a urine sample, divided by plasma creatinine(P_(er)). This is represented as:

C _(er)=(U _(er)×V)/P _(er)=mL/h

The safety parameters monitored during the study were mortality, foodand water intake, and body weight. Mortality checks and cage sideclinical observations were performed once daily throughout the study.Food intake was measured daily and water intake was recorded weekly.Body weights of all animals were recorded at the beginning of the studyand at the end of the study.

As shown in FIG. 3, oral administration of OXDC-CLEC to EG-challengedAGT1 KO (129sv) mice resulted in significant reduction of urinaryoxalate levels from day 4 of the treatment until the end of the studywhen compared with matched untreated control mice. A reduction ofbetween 30 and 50% was observed in all three treatment groups, with themaximal reduction observed in the highest dose group (80 mg ofOXDC-CLEC). Lower closes of 25 mg and 5 mg of OXDC-CLEC producedreduction in urine oxalate up to 35%.

The results are analyzed by unpaired two tail Student's t-test. At thebeginning of the study, each dosage group had n=11 mice, but severalmice died during the course of the study due to the ethylene glycolchallenge. The results presented include only mice that were alive atthe particular day of urine oxalate measurements. The bell shaped curveof urinary oxalate excretion in the control group is best explained bythe observation that initial elevation of urinary oxalate leads tonephrocalcinosis, reduced renal filtering function, and consequentlowered excretion rate of oxalate over time, and eventual death in theworst case.

Assessment of the renal function by creatinine clearance measurement. Atthe end of the study, all animals that survived 4 weeks of EG challengewere sacrificed and blood was collected to measure plasma creatinine andcreatinine clearance. All 11 mice in tire 80 mg dosage group survivedthe 4 weeks of EG challenge; 8 of 11 mice in the 25 mg OXCD-CLEC dosagegroup survived; 8 of 11 mice in the 5 mg OXCD-CLEC dosage groupsurvived; and 7 of 11 mice in the control group survived. For serumcreatinine measurement, the slightly modified Jaffe reaction methoddescribed above was used (see, e.g., the Creatinine Microassay Plate Kitfrom Oxford Medical Research, Inc.; Slot, Scand J. Clin. Lab. Invent.17:381, 1965; and Heinegard. Clin. Chim. Acta 42:305, 1973).

The efficacy of orally administered OXDC-CLEC on kidney function wasassessed by measuring creatinine clearance. Creatinine clearance in themice that survived the entire one month study period is shown in FIG. 4.When compared with the control group, creatinine clearance wassignificantly higher in surviving mice that received 80 mg of OXDC-CLEC(p<0.05).

All mice (11/11) in the 80 mg OXDC-CLEC treatment group survived the 4week EG challenge regimen, while only 7 mice (7/11) in the control groupsurvived the regimen. The kidney filtration rate, assayed by creatinineclearance, was also significantly lower than in the 80 mg dosage group(FIG. 4).

Kidney histopathology analysis: Mouse kidneys were routinely processedfor paraffin embedding and positioned in order to obtain complete crosssections of the kidneys. Each kidney was cut in 12 serial sections at 4μm per kidney and stained with either hemotoxylin and eosin for routinehistological examination, or by specific Yasue metal substitutionhistochemical method to detect the presence of calcium oxalate crystalsin the renal tissue. Slides were examined under the microscope using 20×magnification and examiner scored sections under 4-category scale,applying the same criteria to each of the anatomic areas in the kidney(cortex, medulla and papilla). The scoring was (i) none (no oxalatecrystals in any field); (ii) minimal (1-5 crystals in any field); (iii)moderate (6-10 crystals in any field); and (iv) severe (all fields withmultiple collections of crystals).

Representative images of kidney tissue from both treatment and controlanimals are shown in FIGS. 5A-5C. Yasue-positive calcium oxalatecrystals were visible in the kidney parenchyma at 20× magnification. Allmice treated with 80 mg of OXDC-CLEC treatment group had normal healthykidney with no traces of calcium oxalate deposits (FIG. 5A). Moderatenephrocalcinosis (FIG. 5B) and severe nephrocalcinosis (FIG. 5C wereobserved in the control group and in some mice from low dose treatmentgroups. The white arrows indicate calcium oxalate deposits and the grayarrows in FIG. 5C indicate large area with interstitial fibrosis.

Histological examination of the kidneys using the specific Yasue metalsubstitution method showed deposits primarily in the cortex and medullarpart of the kidneys. In the case of severe nephrocalcinosis (FIG. 5C),calcium oxalate deposits were randomly distributed in the kidneys. Signsof fibrosis and inflammation were also visible and the morphology ofglomeruli was changed with the occasionally formation of calcium oxalatedeposits within the glomeruli. All mice (11/11) from the 80 mg dosagegroup had normal morphology of the kidneys upon necropsy with no tracesof calcium oxalate deposition either in the kidney or in the urinarybladder. In contrast, 100% (11/11) of mice from untreated control grouphad calcium oxalate deposits. In the low treatment groups (25 mg or 5 mgof OXDC-CLEC), 63% (7/11) of mice had calcium oxalate deposits in thekidneys. These results demonstrate a positive, dose dependent effect ofthe oral therapy with OXDC-CLEC in the EG-challenged AGT1 KO mice on thereduction of hyperoxaluria and the prevention of calcium oxalate crystaldeposition in the kidneys. A summary of the histopathological analysisfrom all 4 groups of mice is presented in Table 3.

TABLE 3 Severity of Nephrocalcinosis and Number of Mice Affected inTreated and Control Group Following Oral OXDC-CLEC Treatment. NUMBER OFMICE WITH NEPHROCALCINOSIS CALCIUM OXALATE DOSE MOD- DEPOSITS GROUPS*SEVERE ERATE MINIMAL NONE (%) CONT 4 (4 died) — 7 — 100 n = 11 80 mg — —— 11 0 n = 11 25 mg 3 (3 died) 2 2 4 63 n = 11 5 mg 3 (3 died) — 4 4 63n = 11

The efficacy of the oral OXDC-CLEC treatment on the frequency of urinarystone formation in control mice and mice from three different treatmentgroups was also evaluated. Two main types of calculi were found in theurinary bladder from EG AGT1 KO mice: calcium oxalate monohydrate stonesand calcium oxalate dihydrate stones. Grossly visible bladder stoneswere present in 36% (4/11) of the mice in the control group and in 19%(2/11) of the mice in the two lower treatment groups. No bladder stoneswere observed in the 80 mg high dose OXDC-CLEC group. The X-raydiffraction analysis showed that stones with whitish, rough buddingsurface are mostly composed of calcium oxalate monohydrate, whilerelatively large stones with sharp crystal angles correspond to calciumoxalate dehydrate.

Survival rate analysis by Kaplan-Meier estimator. The effect ofOXDC-CLEC treatment on survival rate of mice challenged with ethyleneglycol was analyzed using the Kaplan-Meier method where survival ofsubjects that died in the certain time point is divided by the number ofsubjects who were still in the study at the time. This methodgraphically illustrates the difference between the groups in the study(FIG. 6). Often statistical programs such as Kaleida graph and STATS areused for calculations.

Oral treatment with OXDC-CLEC increased the survival rate ofEG-challenged AGT1 KO mice as compared to that of the matched controls.All mice (11/11) from the 80 mg OXDC-CLEC treatment group survived the30 day study period without signs of sickness, while 4 of 11 mice fromthe control group had severe nephrocalcinosis with development ofurinary stones.

Since OXDC-CLEC is intended for oral administration, the potential foradverse reactions of the crosslinked crystals on gastrointestinal (GI)tissues was evaluated. The digestive tracts of treated EG-challengedAGT1 KO mice were examined macroscopically and histological analysiswith a hemotoxylin-eosin stain was performed on different parts of theGI tract including the stomach (corpus and antrum) and small bowel(jejunum and ileum). The evaluation confirmed that four weeks of oraltreatment with OXDC-CLEC was well-tolerated and did not cause structuralor morphological changes in the GI tract. Similar results were observed(or the large bowel.

Summary of the dose range study in EG-challenged AGT1 KO mice orallytreated with oxalate decarboxylase-CLEC. Oral treatment with OXDC-CLECin a primary hyperoxaluria animal model was safe and efficacious. Insummary, four weeks of oral treatment with OXDC-CLEC reduced urinaryoxalate by 30-50%. Significant and sustained reduction was recorded witheach of the three doses of the test article evaluated. Four weeks oforal treatment prevented calcium oxalate deposition in the kidneyparenchyma at the highest treatment dose. Four weeks of oral treatmentincreased the survival rate at the two lower doses and prevented animalmortality at the highest dose studied. Finally, the 4 week treatmentregimen, did not produce macroscopic or microscopic changes in the GItract.

Example 15. Assays

Protein Concentration Determination: The concentration of oxalatedecarboxylase was determined by measuring absorbance at 280 nm. Theabsorbance of 1.36 optical density (OD) was considered as 1 mg/ml.

OXDC: Activity Assay: The modified Sigma Aldrich protocol (EnzymaticAssay of Oxalate Decarboxylase EC 4.1.1.2) was used to measure theactivity of soluble oxalate decarboxylase, oxalate decarboxylasecrystals, and cross-linked oxalate decarboxylase crystals (OXDC-CLEC).This is an indirect two-step activity assay; in the first reaction OXDCconverts the substrate oxalate to formate and carbon dioxide. In thesecond reaction, formate hydrogen is stochiometrically transferred toNAD to form NADH by formate dehydrogenase. The ensuing concentration ofNADH is quantified spectrophotomerically at 340 nm. Unit (u) ofenzymatic activity is defined as follows: one Unit of oxalatedecarboxylase will form 1.0 μmol of oxalate to formate and carbondioxide per minute at pH 5 and 37° C.

Assay samples were normalized at a concentration of 0.007-0.02 and0.009-0.03 mg/mL for OXDC crystals and OXDC-CLEC, respectively, in 5 mMpotassium phosphate buffer pH 7.0 with 1 mM DTT (Sigma). Proteinconcentrations were determined by absorbance at 280 nm. Formatedehydrogenase (FDH) at 40 U/mL was prepared in cold diH₂O prior to useand kept on ice. All other reagents were kept at room temperature.Reagents were added to 2 ml microtubes with stir bar in the followingorder: 300 μL of 100 mM potassium phosphate and 200 μL of potassiumoxalate pH 4.0 were mixed and warmed for 5 min in a water bath at 37°C., then 100 μL, of 5 mM potassium phosphate with 1 mM DTT was added tothe blank vial and 100 μL of diluted oxalate decarboxylase was added toall other vials. After 2 minutes, the reaction was stopped with 130 mMpotassium phosphate dibasic. In the second reaction, 25 μL of NADsolution was added to 100 μL of FDH solution, and the incubation wascontinued for another 20 min. All samples were then centrifuged for 1min at 16,100 rpm. Reaction mixtures were then transferred to 1.5 mL UVcuvettes, and absorbance at 340 nm was determined and recorded using aShimadzu BioSpec (Shimadzu Scientific Instruments, Columbia, Md.).

Enzyme Specific Activity was Calculated as Follows:

${{Units}\text{/}{mL}\mspace{14mu} {OXDC}} = \frac{\left\lbrack {{Abs} \times {Total}\mspace{14mu} {volume}\mspace{14mu} \left( {1.725\mspace{14mu} {mL}} \right) \times {{dil}.\mspace{14mu} {factor}}} \right\rbrack}{\begin{matrix}{\left( {{{Ext}.\mspace{14mu} {NADH}} = 6.22} \right)\left( {{{Vol}.\mspace{14mu} {OXDC}} = {0.1\mspace{14mu} {mL}}} \right)} \\\left( {{{assay}\mspace{14mu} {time}} = {2\text{/}5\mspace{14mu} \min}} \right)\end{matrix}}$

In a specific experiment, cross-linked crystals of oxalate decarboxylase(OXDC-CLEC) (diamond shaped crystals cross-linked with 1% glutaraldehydeby tumbling overnight; see Example 9) were compared to OXDC crystals.OXDC-CLEC retained 50% of the activity of the corresponding crystallineOXDC preparation.

Oxalate determination by colorimetric method: Oxalate colorimetric kitfor quantitative determination of oxalate in the urine were purchasedfrom Trinity Biotech USA (St. Louis, Mo.) or Greiner Diagnostic AG(Dennliweg 9, Switzerland). The urine samples were diluted and treatedaccording to the manufacturer's instruction. The assay comprises twoenzymatic reactions: (a) oxalate is oxidized to carbon dioxide andhydrogen peroxide by oxalate oxidase, and (b) the hydrogen peroxide thusformed reacts with 3-methyl-2-benzothiazolinone hydrazone (MBTH) and3-(dimethylamino)benzoic acid (DMAB) in the presence of peroxidase toyield an indamine dye which can be detected by absorbance at 590 nm. Theintensity of the color produced is directly proportional to theconcentration of oxalate in the sample. Urine oxalate values arecalculated from standard curve.

Creatinine determination by colorimetric method: Creatinine colorimetrickits for the quantitative determination of creatinine in the urine werepurchased from Quidel Corporation (San Diego, Calif.: METRA CreatinineAssay kit) or Randox Laboratories (Antrim, United Kingdom). The assay isbased on the principle that creatinine reacts with picric acid inalkaline solution to form a product that has an absorbance at 492 nm.The amount of complex formed is directly proportional to the creatinineconcentration. 24 h rat urine samples collected from single metaboliccages were diluted 15 fold with double distilled water. 20 μl of dilutedurine sample was mixed with 20 μl picric acid/sodium hydroxide (1:1).Absorbance at 492 nm was measured after incubating for 2 minutesincubation at room temperature. Urinary creatinine values werecalculated from standard curve.

Example 16. OXDC Therapy for the Treatment of Oxalate-AssociatedDisorders in Humans

Humans in need of treatment or prevention of an oxalate-associateddisorder such as hyperoxaluria can be treated by oral administration ofcross-linked oxalate decarboxylase crystals. The oxalate decarboxylasecrystals are administered at an approximate dose of 10 μg/kg to 25mg/kg; 1 mg/kg to 25 mg/kg, or 5 mg/kg to 100 mg/kg, as determined by atreating clinician and depending on the severity of the symptoms and theprogression of the disease. The oxalate decarboxylase crosslinkedcrystals are administered 1, 2, 3, 4, or 5 times daily, or areadministered less frequently, such as once or twice a week. This oraladministration of OXDC-CLEC results in a decrease in urinary oxalatelevels of at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%or more.

Example 17. Recombinant Production of Oxalate Decarboxylase

In Human Embryonic Kidney (HEK293) cells: DMA encoding OXDC (e.g., SEQID NO: 1 or 2) is cloned into a suitable expression vector. Aftersequence confirmation, the vector can be linearized and transformationof the linearized vector to pre-seeded HEK293 cells may be carried outusing Lipofectamine™ 2000 Transfection Reagent in a 6 cm diameter dish.The transfection reaction is cultured overnight in appropriate medium,and then transformants are selected in medium supplemented with 0.5 g/Lof neomycin. Stably transfected HEK293 cell clones are identified aftergrowth in neomycin-containing medium for up to 3 weeks. The clones arethen isolated and propagated, and used for OXDC expression.

In Chinese Hamster Ovary (CHO) cells: DNA encoding OXDC gene is clonedinto a suitable expression vector. Cultured CHO lec 3.2.8.1 cells arethen detached by trypsin digestion and harvested by centrifugation. Thecells are then suspended in Electroporation phosphate buffered salinebutter (EPBS) to a final concentration of ˜1×10⁷/ml, and transformedwith the linearized vector by electroporation. After overnight culture,the medium is exchanged with medium supplemented with 0.5 g/L neomycin.Successive changes of medium are performed to screen for stablytransfected CHO cell clones. Once the stably transfected cell clones areestablished and propagated, the cells are used for OXDC expression.

In Pichia Pastoris: DNA encoding the OXDC gene is cloned into a suitableexpression vector. After sequence confirmation, the vector can belinearized then transformed into a Pichia Pastoris host cell (see,Whittaker et al. J. Biol. Inorg, Chem. 7:136-145, 2002). Transformantsare selected with Zeocin, expanded in buffered glycerol complex medium(BMGY), and induced with methanol. OXDC may then be isolated from theculture medium.

In Saccharomyces cerevisiae: The synthetic OXDC, gene can be cloned intoa suitable expression vector containing, for example, the Gal1 promoter(pGal) and terminator sequences for expression. After sequenceconfirmation, the expression vector is transformed into the competentSaccharomyces cerevisiae W303-1A by electroporation. The transformantsare screened and propagated before use for OXDC expression.

In insect cells: DNA encoding OXDC may be cloned into a suitableexpression vector, such as, e.g., a baculovirus system. After sequenceconfirmation, the vector may be transformed into competent DH10Bac E.coli cells, and E. coli cells containing the recombinant bacmid can bescreened and verified. The recombinant bacmid DNA is isolated and usedto transfect insect Sf9 cells using reagents such as Cellfectin™ reagent(Invitrogen, Carlsbad, Calif.). The recombinant baculovirus particlescan then be isolated, propagated, and titered before use to infect Sf9cells for OXDC expression. In E. coli: DNA encoding OXDC is cloned intoa suitable E. coli expression vector. After sequence confirmation, thevector is transformed into competent E. coli BL21 or if necessary E.coli Origami B (DE3), which allows the formation of disulfide bonds inthe recombinant protein expressed in this strain. The transformants arescreened by growing the transformants on nutrient plates containingantibiotics and verified by colony HCR using OXDC gene specific primers.The transformants are then cultured in the liquid medium and inducedwith isopropyl-beta-D-thiogalactopyranoside (IPTG) for OXDC expression.

Example 18. Sequences

Collybia velutipes sequence (SEQ ID NO:1) to express in Candidaboidinii. Two Not I sequences are underlined; the ATA triplet in bold isa spacer codon; the double-underlined sequence is alpha mating factorsequence optimized for C. Boidinii; the OXDC coding sequence is depictedin lower-case letters.

   1

  50 TTATTTGCTGCTTCTTCTGCTTTAGCTGCTCCAGTTAATACTACTACTGA  100AGATGAAACTGCTCAAATTCCAGCTGAAGCTGTTATTGGTTATTCTGATT  150TAGAAGGTGATTTTGATGTTGCTGTTTTACCATTTTCTAATTCTACTAAT  200AATGGTTTATTATTTATTAATACTACTATTGCTTCTATTGCTGCTAAAGA  250AGAAGGTGTTTCTTTAGAAAAAAGAGAAGCTGAAGCTatgtttaataatt  300ttcaaagattattaactgttattttattatctggttttactgctggtgtt  350ccattagettctactactactggtactggtactgctactggtacttctac  400tgctgctgaaccatctgctactgttccatttgcttctactgatccaaatc  450cagttttatggaatgaaacttctgatccagctttagttaaaccagaaaga  500aatcaattaggtgctactattcaaggtccagataatttaccaattgattt  550acaaaatccagatttattagctccaccaactactgatcatggttttgttg  600gtaatgctaaatggccattttctttttctaaacaaagattacaaactggt  650ggttgggctagacaacaaaatgaagttgttttaccattagctactaattt  700agcttgtactaatatgagattagaagctggtgctattagagaattacatt  750ggcataaaaatgctgaatgggcttatgttttaaaagggtctactcaaatt  800tctgctgttgataatgaagggagaaattatatttctactgttggtccagg  850tgatttatggtattttccaccaggcattccacattctttacaagctactg  900ctgatgatccagaaggttctgaatttattttagtttttgattctggtgct  950tttaatgatgatggtacttttttattaactgattggttatctcatgttcc 1000aatggaagttattttaaaaaattttagagctaaaaatccagctgcttggt 1050ctcatattccagctcaacaattatatatttttccatctgaaccaccagct 1100gataatcaaccagatccagtttctccacaagggactgttccattaccata 1150ttcttttaatttttcttctgttgaaccaactcaatattctggtgggactg 1200ctaaaattgctgattctactacttttaatatttctgttgctattgctgtt 1250gctgaagttactgttgaaccaggtgctttaagagaattacattggcatcc 1300aactgaagatgaatggactttttttatttctggtaatgctagagttacta 1350tttttgctgctcaatctgttgcttctacttttgattatcaaggtggtgat 1400attgcttatgttccagcttctatgggtcattatgttgaaaatattggtaa 1450tactactttaacttatttagaagtttttaatactgatagatttgctgatg 1500tttctttatctcaatggttagctttaactccaccatctgttgttcaagct 1550catttaaatttagatgatgaaactttagctgaattaaaacaatttgctac 1600taaagctactgttgttggtccagttaattaaGCGGCCGCtaaactat 1646Bacillus subtilis sequence (SEQ ID NO:2): The underlined “G” at position705 indicates an A→G base substitution. This base substitution does notalter the amino acid sequence.

   1  ATGAAAAAACAAAATGACATTCCGCAGCCAATTAGAGGAGACAAAGGAG   50CAACGGTAAAAATCCCGCGCAATATTGAAAGAGACCGGCAAAACCCTGAT  100ATGCTCGTTCCGCCTGAAACCGATCATGGCACCGTCAGCAATATGAAGTT  150TTCATTCTCTGATACTCATAACCGATTAGAAAAAGGCGGATATGCCCGGG  200AAGTGACAGTACGTGAATTGCCGATTTCAGAAAACCTTGCATCCGTAAAT  250ATGCGGCTGAAGCCAGGCGCGATTCGCGAGCTTCACTGGCATAAAGAAGC  300TGAATGGGCTTATATGATTTACGGAAGTGCAAGAGTCACAATTGTAGATG  350AAAAAGGGCGCAGCTTTATTGACGATGTAGGTGAAGGAGACCTTTGGTAC  400TTCCCGTCAGGCCTGCCGCACTCCATCCAAGCGCTGGAGGAGGGAGCTGA  450GTTCCTGCTCGTGTTTGACGATGGATCATTCTCTGAAAACAGCACGTTCC  500AGCTGACAGATTGGCTGGCCCACACTCCAAAAGAAGTCATTGCTGCGAAC  550TTCGGCGTGACAAAAGAAGAGATTTCCAATTTGCCTGGCAAAGAAAAATA  600TATATTTGAAAACCAACTTCCTGGCAGTTTAAAAGATGATATTGTGGAAG  650GGCCGAATGGCGAAGTGCCTTATCCATTTACTTACCGCCTTCTTGAACAA  700GAGCCGATCGAATCTGAGGGAGGAAAAGTATACATTGCAGATTCGACAAA  750CTTCAAAGTGTCTAAAACCATCGCATCAGCGCTCGTAACAGTAGAACCCG  800GCGCCATGAGAGAACTGCACTGGCACCCGAATACCCACGAATGGCAATAC  850TACATCTCCGGTAAAGCTAGAATGACCGTTTTTGCATCTGACGGCCATGC  900CAGAACGTTTAATTACCAAGCCGGTGATGTCGGATATGTACCATTTGCAA  950TGGGTCATTACGTTGAAAACATCGGGGATGAACCGCTTGTCTTTTTAGAA 1000ATCTTCAAAGACGACCATTATGCTGATGTATCTTTAAACCAATGGCTTGC 1050CATGCTTCCTGAAACATTTGTTCAAGCGCACCTTGACTTGGGCAAAGACT 1100TTACTGATGTGCTTTCAAAAGAAAAGCACCCAGTAGTGAAAAAGAAATGC 1150 AGTAAATAA 1158

The translated oxalate decarboxylase protein from Bacillus subtilissequence is shown below (Swiss-Prot: O34714) (SEQ ID NO:3).

   1  MKKQNDIPQPIRGDKGATVKIPRNIERDRQNPDMLVPPETDHGTVSNMK   50FSFSDTHNRLEKGGYAREVTVRELPISENLASVNMRLKPGAIRELHWHKE  100AEWAYMIYGSARVTIVDEKGRSFIDDVGEGDLWYFPSGLPHSIQALEEGA  150EFLLVFDDGSFSENSTFQLTDWLAHTPKEVIAANFGVTKEEISNLPGKEK  200YIFENQLPGSLKDDIVEGPNGEVPYPFTYRLLEQEPIESEGGKVYIADST  250NFKVSKTIASALVTVEPGAMRELHWHPNTHEWQYYISGKARMTVFASDGH  300ARTFNYQAGDVGYVPFAMGHYVENIGDEPLVFLEIFKDDHYADVSLNQWL  350AMLPETFVQAHLDLGKDFTDVLSKEKHPVVKKKCSK  385

Example 19. Stability of Soluble OXDC Crystalline OXDC and OXPC-CLEC atLow pH 3.0

Soluble OXDC, 2.5 mg/mL (Example 5), crystalline OXDC, 5.0 mg/mL(Example 5) and OXDC-CLEC, 5.0 mg/mL (Example 10) were taken in sodiumcitrate buffer pH 3.0, 1 mL each in Eppendorf tubes and incubated for 5h at 37° C. Samples were taken at 0, 2 and 5 hrs for measurement ofstability of the enzyme. The activities were determined as in Example15. Results showing the stability of OXDC and OXDC-CLEC at pH 3.0 after0.2. and 5 hrs are shown in FIG 7. OXDC-CLEC retained about 100%activity after 5h of incubation at pH 3.0, while soluble OXDC withinfirst 2 h lost about 51% activity and after 5 h had only about 40%activity remained when compared with original. The crystalline OXDC wasmore stable than the soluble OXDC by retaining about 68% activity in 1hr and about 67% activity in 2 hr.

Example 20. Stability of Soluble OXDC, Crystalline OXDC, and OXDC-CLECCrystals in the Presence of Pensin

Soluble OXDC, 1.0 mg/mL (Example 5), crystalline OXDC, 10.0 mg/mL(Example 5) and OXDC-CLEC, 1.0 mg/mL (Example 10) were taken in sodiumcitrate buffer pH 3.0, 1 mL each in Eppendorf tubes and incubated withpepsin (pepsin stock was made at a concentration of 1 mg/mL in 25 mMTris-HCL buffer, pH 7.5) at a ratio of 50:1 between OXDC to pepsin for 5h at 37° C. Samples were taken at 0, 2 and 5 hrs for measurement ofstability of the enzyme. The activities were determined as in Example15. Results showing the stability of soluble OXDC, crystalline OXDC andOXDC-CLEC in the presence of pepsin after 0, 2, and 5 hrs are depictedin FIG. 8.

As shown in FIG. 8, superiority of the OXDC-CLEC formulation versussoluble OXDC was revealed at both low pH and in the presence of pepsin.OXDC-CLEC sample retained about 60% activity after 5 h incubation at 37°C., while majority of soluble OXDC and uncrosslinked crystalline OXDCwere degraded after 2 h by pepsin and only about 20% activity remainedafter 5 h.

Example 21. Stability of Soluble OXDC, Crystalline OXDC, and OXDC-CLECin the Presence of Chymotrypsin

Soluble OXDC, 1.0 mg/mL (Example 5), crystalline OXDC, 10.0 mg/mL(Example 5) and OXDC-CLEC, 1.0 mg/mL (Example 10) were taken in 25 mMTris-HCl buffer at pH 7.5, 1 mL each in Eppendorf tubes and incubatedwith chymotrypsin (chymotrypsin stock was made at a concentration of 1mg/mL in 25 mM Tris-HCl buffer, pH 7.5, freshly prepared) at a ratio of50:1 between OXDC to chymotrypsin for 5 h at 37° C. Samples were takenat 0, 2 and 5 hours for measurement of stability of the enzyme. Theactivities were determined as in Example 15. Results showing thestability of soluble OXDC, crystalline OXDC and OXDC-CLEC in thepresence of chymptrypsin at 0, 2 and 5 hours are shown in FIG 9. Resultsfrom FIG. 9 show that OXDC-CLEC and uncrosslinked crystalline OXDC werestable and resistant to proteolytic cleavage by chymotrypsin after 5hours of incubation at 37° C. Analysis showed that OXDC-CLEC anduncrosslinked crystalline OXDC enzyme retained about 100% activity whencompared to its original values in 5 hours. At the same time, solubleprotein lost about 50% activity after 2 hours exposure to chymotrypsinand 5 hours later all soluble OXDC was degraded, with 0% activityremained.

Example 22. Stability of Soluble OXDC, crystalline OXDC, and OXDC-CLECin Soluble OXDC, 5.0 mg/mL (Example 5), crystalline OXDC, 10.0 mg/mL

(Example 5) and OXDC-CLEC, 10.0 mg/mL (Example 10) were taken insimulated intestinal juice (simulated intestinal juice made according toUSB recommendation. 6.8 gm of monobasic potassium phosphate wasdissolved in 250 ml of water, mixed with 77 ml 0.2 N of sodium hydroxideand 500 ml of deionized water; then, 10 gm of pancreatin was added andpH was adjusted with either 0.2N hydrochloric acid or 0.2 N sodiumhydroxide to a pH of 6.8; water was added to 1L), 1 mL each, inEppendorf tubes and incubated for 2 h at 37° C. Samples were taken at 0,1 and 2 hrs for measurement of stability of the enzyme. The activitieswere determined as in Example 15. The results are shown in FIG. 10.

The results, shown in FIG. 10 indicate that OXDC-CLEC is stable insimulated intestinal juice with pancreatin with about 100% activitypreserved. In contrast, the majority of soluble OXDC was degraded bypancreatin (mix of lipase, amylase and protease) within 1 hour and onlybetween about 26%-28% activity remained after 1 h and 2 h incubation,respectively. The uncrosslinked crystalline OXDC was much more stablethan its soluble form in the presence of pancreatin where it lost onlyabout 76% and about 49% activities after 1 and 2 hr incubation,respectively.

In addition, OXDC-CLEC protects the enzyme from cleavage from proteases,such as pepsin and chymotrypsin. Stability of OXDC-CLEC can be at leastabout 100%, 200%, 300%, 400% or more of that of the stability of solubleOXDC in the same conditions. OXDC-CLEC maintains its activities at leastabout 2, 3, or 4 folds higher than soluble OXDC maintaining itsactivities under the same conditions. Thus, compared to soluble OXDC,OXDC-CLEC is both active and stable in the severe conditions of the gut,ranging from acidic pH of about 2.5 or 3 to pH of about 7.5 or 8.5 andcontaining various proteases.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. A pharmaceutical composition comprising an oxalate decarboxylasecrystal. 2.-33. (canceled)